Isolated nucleic acid encoding corticotropin-releasing factor2 receptors

ABSTRACT

The present invention provides isolated nucleic acid molecules encoding CRF 2  receptors, recombinant expression vectors and host cells suitable for expressing such receptors, as well as compositions and methods which utilize such receptors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application U.S. Ser. No. 08/259,959, filed Jun. 14, 1994, now abandoned.

TECHNICAL FIELD

The present invention relates generally to cell surface receptors, and more specifically, to Corticotropin-Releasing Factor₂ receptors.

BACKGROUND OF THE INVENTION

Corticotropin-releasing factor ("CRF") is a 41-amino acid peptide originally isolated from the hypothalamus by virtue of its ability to stimulate the production of adrenocorticotropic hormone ("ACTH") and other proopiomelanocortin ("POMC") products of the anterior pituitary (Vale et al., Science 213:1394-1397, 1981). Briefly, CRF is believed to initiate its biological effects by binding to a plasma membrane receptor which is distributed throughout the brain (DeSouza et al., Science 224:1449-1451, 1984), pituitary (Wynn et al., Biochem. Biophys. Res. Comm. 110:602-608, 1983), adrenals (Udelsman et al., Nature 319:147-150, 1986) and spleen (Webster, E. L., and E. B. DeSouza, Endocrinology 122:609-617, 1988). This receptor is coupled to a GTP-binding protein (Perrin et al., Endocrinology 118:1171-1179, 1986) which mediates CRF-stimulated increase in intracellular cAMP (Bilezikjian, L. M., and W. W. Vale, Endocrinology 113:657-662, 1983).

In addition to its role in stimulating the production of ACTH and POMC, CRF is also believed to coordinate many of the endocrine, autonomic, and behavioral responses to stress, and may be involved in the pathophysiology of affective disorders. Moreover, CRF is believed to be a key intermediary in communication between the immune, central nervous, endocrine and cardiovascular systems (Crofford et al., J. Clin. Invest. 90:2555-2564, 1992; Sapolsky et al., Science 238:522-524, 1987; Tilders et al., Regul. Peptides 5:77-84, 1982; Fisher et al., Reg. Peptide 5:153-161, 1983).

A receptor for CRF has been cloned from rat (Perrin et al., Endo 133(6):3058-3061, 1993), and human brain (Chen et al., PNAS 90(19):8967-8971, 1993; Vita et al., FEBS 335(1):1-5, 1993). This receptor is a 415 amino acid protein comprising seven membrane spanning domains, and has a predicted molecular weight of 44,000 daltons. A comparison of identity between rat and human sequences shows a high degree of homology (97%) at the amino acid level. In addition, Scatchard analysis of recombinantly produced human receptor demonstrates a single component site, with a high affinity (K_(d) of 1.6±0.3 nM) for CRF.

The present invention provides new, previously unidentified CRF receptors, designated as the "Corticotropin-Releasing Factor-₂ ("CRF₂ "), Corticotropin-Releasing Factor receptors. In addition, the present invention provides compositions and methods which utilize such CRF₂ receptors, as well as other, related advantages.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides compositions and methods which utilize CRF₂ receptors (also termed "CRF₂ Corticotropin-Releasing Factor receptors" or "CRF₂ R"). Within one aspect of the present invention, isolated nucleic acid molecules are provided which encode CRF₂ receptors. Within one embodiment, nucleic acid molecules are provided which encode a CRF₂ receptor such as that disclosed in Sequence I.D. No. 4, from amino acid number 1 to amino acid number 411. Within another embodiment, nucleic acid molecules are provided which comprise the sequence of nucleotides in Sequence I.D. No. 3, from nucleotide number 216 to nucleotide number 1449. Within another embodiment, nucleic acid molecules are provided which encode a CRF₂ receptor such as that disclosed in Sequence ID No. 2 from amino acid number 1 to amino acid number 431. Within another embodiment, nucleic acid molecules are provided which comprise the sequence of nucleotides in Sequence ID No. 1 from nucleotide number 44 to nucleotide number 1336. Nucleic acid molecules which encode CRF₂ receptors of the present invention may be isolated from virtually any warm-blooded animal, including for example, humans, macaques, horses, cattle, sheep, pigs, dogs, cats, rats and mice.

Within another aspect of the present invention, isolated nucleic acid molecules are provided which encode portions of a CRF₂ receptor, such as the N-terminal extracellular domain. Within one embodiment, isolated nucleic acid molecules are provided comprising the sequence of nucleotides in Sequence I.D. No. 3, from nucleotide number 216 to nucleotide number 570. Within another embodiment, isolated nucleic acid molecules are provided which encode a protein having the amino acid sequence of Sequence I.D. No. 4, from amino acid number 1 to amino acid number 118. Within another embodiment, isolated nucleic acid molecules are provided comprising the sequence of nucleotides in Sequence ID No. 1 from nucleotide number 44 to nucleotide number 451. Within another embodiment, isolated nucleic acid molecules are provided which encode a protein having the amino acid sequence of Sequence ID No. 2 from amino acid number 1 to amino acid number 138.

Within other aspects of the invention, expression vectors are provided which are capable of expressing the above-described nucleic acid molecules. Within other aspects, recombinant viral vectors are provided which are capable of directing the expression of the above-described nucleic acid molecules. Representative examples of such viral vectors include retroviral vectors, adenoviral vectors, and herpes simplex virus vectors. Also provided by the present invention are host cells which contain the above-described expression vectors, as well as the receptor or portions thereof which are encoded by the above-described nucleic acid molecules. Within other embodiments, isolated portions of CRF₂ receptors are provided, including for example, isolated portions of extracellular domains such as the N-terminal extracellular domain.

Within other aspects of the invention, isolated antibodies are provided which are capable of specifically binding to the above-described CRF₂ receptors. Within one embodiment, the antibodies may be selected from the group consisting of polyclonal antibodies, monoclonal antibodies, and antibody fragments. Within other embodiments, antibodies are provided which are capable of blocking the binding of CRF (or other substrates such as sauvagine or urotensin I) to a CRF₂ receptor. Within preferred embodiments, the antibodies may be selected from the group consisting of murine and human antibodies. Within preferred aspects of the invention, the above-noted antibodies are produced by hybridomas.

Within yet another aspect of the present invention, nucleic acid molecules are provided which are capable of specifically hybridizing to a nucleic acid molecule encoding any of the CRF₂ receptors described above. Such molecules may be between at least "y" nucleotides long, wherein "y" is any integer between 14 and 1230, and furthermore, may be selected suitable for use as probes or primers described below. Particularly preferred probes of the present invention are at least 18 nucleotides in length.

Within other aspects of the present invention, methods for detecting the presence of a compound which binds to a CRF₂ receptor are provided, comprising the steps of (a) exposing one or more compounds to cells that express CRF₂ receptors under conditions and for a time sufficient to allow binding of the compounds to the receptors, and (b) isolating compounds which bind to the receptors, such that the presence of a compound which binds to a CRF₂ receptor may be detected. Within another aspect, methods for detecting the presence of a compound which binds to a CRF₂ receptor are provided, comprising the steps of (a) exposing one or more compounds to a CRF₂ receptor N-terminal extracellular domain under conditions and for a time sufficient to allow binding of a compound to the N-terminal extracellular domain, and (b) isolating compounds which bind to the CRF₂ receptor N-terminal extracellular domain, such that the presence of a compound which binds to a CRF₂ receptor may be detected. Within one embodiment, the compounds are labeled with an agent selected from the group consisting of fluorescent molecules, enzymes, and radionuclides.

Within other aspects of the present invention, methods for determining whether a selected compound is a CRF₂ receptor agonist or antagonist are provided, comprising the steps of (a) exposing a selected compound to cells which express CRF₂ receptors under conditions and for a time sufficient to allow binding of the compound and an associated response in intracellular levels of cAMP, and (b) detecting either an increase or decrease in the level of intracellular cAMP, and thereby determining whether the selected compound is a CRF₂ receptor agonist or antagonist.

Within other aspects, methods are provided for detecting the presence of a CRF₂ receptor agonist or antagonist in a pool of compounds, comprising the steps of (a) exposing a pool of compounds to cells which express CRF₂ receptors under conditions and for a time sufficient to allow binding of the compound and an associated response in intracellular levels of cAMP, and (b) isolating compounds which either increase or decrease the intracellular level of cAMP, such that the presence of a CRF₂ receptor agonist or antagonist may be detected.

Within another aspect, methods for determining whether a selected compound is a CRF₂ receptor antagonist are provided, comprising the steps of (a) exposing a selected compound in the presence of a CRF₂ receptor agonist to a recombinant CRF₂ receptor coupled to a response pathway under conditions and for a time sufficient to allow binding of the compound to the receptor and an associated response through the pathway, and (b) detecting a reduction in the stimulation of the response pathway resulting from the binding of the compound to the CRF₂ receptor, relative to the stimulation of the response pathway by the CRF₂ receptor agonist alone, and therefrom determining the presence of a CRF₂ antagonist. Within other aspects, methods are provided for determining whether a selected compound is a CRF₂ receptor agonist, comprising the steps of (a) exposing a selected compound to a recombinant CRF₂ receptor coupled to a response pathway under conditions and for a time sufficient to allow binding of the compound to the receptor and an associated response through the pathway, and (b) detecting an increase in stimulation of the response pathway resulting from the binding of the compound to the CRF₂ receptor, and therefrom determining the presence of a CRF₂ receptor agonist.

These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are set forth below which describe in more detail certain procedures or compositions (e.g., plasmids, etc.), and are therefore incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates the structure of one representative CRF₂ receptor (CRF₂β ; Sequence ID No. 2).

FIG. 2 schematically illustrates the structure of a second representative CRF₂ receptor (CRF₂α ; Sequence ID No. 4).

FIG. 3 is a graph which depicts cAMP accumulation in cells transfected with the CRF₁ receptor.

FIG. 4 is a graph which depicts cAMP accumulation in cells transfected with the CRF₂ receptor.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Prior to setting forth the invention, it may be helpful to an understanding thereof to set forth definitions of certain terms to be used hereinafter.

"CRF₂ receptors" (also termed "CRF₂ Corticotropin-Releasing Factor receptors" or "CRF₂ R") as utilized herein refers to receptor proteins which bind corticotropin-releasing factor and other proteins such as urotensin I and sauvagine. CRF₂ receptors may be distinguished from other receptors such as Corticotropin-Releasing Factor receptors based upon criteria such as affinity of substrate binding, tissue distribution, and sequence homology. For example, CRF₂ receptors of the present invention should be greater than 70% homologous, preferably greater than 75% to 80% homologous, more preferably greater than 85% to 90% homologous, and most preferably greater than 92%, 95%, or 97% homologous to the CRF₂ receptors disclosed herein (e.g., Sequence I.D. No. 3). In their native configuration, CRF₂ receptors are believed to exist as membrane bound proteins, consisting of an N-terminal extracellular domain, seven transmembrane domains separated by three intracellular and three extracellular loops, and a C-terminal intracellular domain (see FIGS. 1 and 2). As utilized within the context of the present invention CRF₂ receptors should be understood to include not only the proteins which are disclosed herein (see Sequence I.D. Nos. 2, 4 and 8), but substantially similar derivatives and analogs as discussed below.

"Nucleic acid molecule" refers to a nucleic acid sequence, in the form of a separate fragment or as a component of a larger nucleic acid construct, which has been derived from nucleic acid isolated at least once in substantially pure form, i.e., free of contaminating endogenous materials, and in a quantity or concentration enabling identification, manipulation, and recovery of the sequence and its component nucleotide sequences by standard biochemical methods, for example, using a cloning vector. Such sequences are preferably provided in the form of an open reading frame uninterrupted by internal nontranslated sequences, or introns, which are typically present in eukaryotic genes. Genomic nucleic acid containing the relevant sequences may also be used. Sequences of non-translated nucleic acid may be present 5' or 3' from the open reading frame, where the same do not interfere with manipulation or expression of the coding regions.

"Recombinant expression vector" refers to a replicable nucleic acid construct used either to amplify or to express nucleic acid sequences which encode CRF₂ receptors. This construct comprises an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences.

As noted above, the present invention provides isolated nucleic acid molecules encoding CRF₂ receptors. Briefly, CRF₂ receptors are G-coupled protein receptors, which are capable of binding a substrate (such as CRF, or other substrates such as sauvagine and urotensin I), and transducing the signal provided by the substrate to the cell. Such signal transduction typically occurs when a response pathway is activated by an external stimulus that is generally, but not always, directly coupled to a membrane-bound receptor. Response pathways generally induce cellular responses such as extracellular matrix secretion from responsive cell lines, hormone secretion, chemotaxis, differentiation, or the initiation or inhibition of cell division of responsive cells. As used herein, the coupling of receptors to response pathways refers to the direct activation of a response pathway or the transduction of a signal via a second messenger, such as a G-protein, to activate a cellular response pathway.

One representative CRF₂ receptor which may be obtained utilizing the methods described herein (see Example 1) is schematically illustrated in FIG. 1 (see also FIG. 2. Briefly, this CRF₂ receptor is composed of an Extracellular N-terminal Domain (amino acids 1-117), a first Transmembrane Domain (amino acids 118-138), a first Intracellular Domain (139-147), a second Transmembrane Domain (148-167), a second Extracellular Domain (168-184), a third Transmembrane Domain (185-208), a second Intracellular Domain (229-223), a fourth Transmembrane Domain (224-244), a third Extracellular Domain (245-261), a fifth Transmembrane Domain (262-286), a third Intracellular Domain (287-309), a sixth Transmembrane Domain (310-329), a fourth Extracellular Domain (330-342), a seventh Transmembrane Domain (343-363), and a C-terminal Intracellular Domain (364-411).

Although the above CRF₂ receptor has been provided for purposes of illustration (see also FIG. 2 and Sequence I.D. No. 2), the present invention should not be so limited. In particular, the present invention provides a wide variety of additional CRF₂ receptors which have substantial similarity to the sequences disclosed in Sequences I.D. Nos. 1-4. As utilized within the context of the present invention, nucleic acid sequences which encode CRF₂ receptors are deemed to be substantially similar to those disclosed herein if: (a) the nucleic acid sequence is derived from the coding region of a native CRF₂ receptor gene (including, for example, allelic variations of the sequences disclosed herein); (b) the nucleic acid sequence is capable of hybridization to nucleic acid sequences of the present invention under conditions of either moderate (e.g. 50% formamide, 5×SSPE, 5×Denhardt's, 0.1% SDS, 100 ug/ml Salmon Sperm nucleic acid, and a temperature of 42° C.) or high stringency (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, NY, 1989); or (c) nucleic acid sequences are degenerate as a result of the genetic code to the nucleic acid sequences defined in (a) or (b). Furthermore, although nucleic acid molecules are primarily referred to herein, as should be evident to one of skill in the art given the disclosure provided herein, a wide variety of related nucleic acid molecules may also be utilized in various embodiments described herein, including for example, RNA, nucleic acid analogues, as well as chimeric nucleic acid molecules which may be composed of more than one type of nucleic acid.

In addition, as noted above, within the context of the present invention "CRF₂ receptors" should be understood to include derivatives and analogs of the CRF₂ receptors described above. Such derivatives include allelic variants and genetically engineered variants that contain conservative amino acid substitutions and/or minor additions, substitutions or deletions of amino acids, the net effect of which does not substantially change the biological activity (e.g., signal transduction) or function of the CRF₂ receptor. Such derivatives are generally greater than about 70% to 75% similar to the corresponding native CRF₂ receptor, preferably greater than 80% to 85% similar, more preferably greater than 90% to 95% similar, and most preferably greater than 97% similar. Percent similarity may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), as revised by Smith and Waterman (Adv. Appl. Math. 2:482, 1981). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences. The preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess (Nucl. Acids Res. 14:6745, 1986), as described by Schwartz and Dayhoff (ed., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.

The primary amino acid structure of CRF₂ receptors may also be modified by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like, or by creating amino acid sequence mutants. Covalent derivatives may be prepared by linking particular functional groups of CRF₂ R amino acid side chains or at the N- or C-termini. Other derivatives of CRF₂ R within the scope of this invention include covalent or aggregative conjugates of CRF₂ R or its fragments with other proteins or polypeptides, such as by synthesis in recombinant culture as N-terminal or C-terminal fusions. For example, the conjugated peptide may be a signal (or leader) polypeptide sequence at the N-terminal region of the protein which co-translationally or post-translationally directs transfer of the protein from its site of synthesis to its site of function inside or outside of the cell membrane or wall (e.g., the yeast α-factor leader). CRF₂ R protein fusions may also comprise peptides added to facilitate purification or identification of CRF₂ R (e.g., poly-His). More specifically, a fusion construct such as human CRF₂ R (His)_(n) or sCRF₂ R (His)_(n) may be constructed in order to allow purification of the protein via the poly-His residue, for example, on a NTA nickel-chelating column. The amino acid sequence of a CRF₂ receptor may also be linked to the Flag peptide (Hopp et al., Bio/Technology 6:1204, 1988) in order to facilitate purification of expressed recombinant protein.

The present invention also includes CRF₂ R proteins with or without associated native-pattern glycosylation. Briefly, CRF₂ R expressed in yeast or mammalian expression systems, e.g., COS-7 cells, may be similar or significantly different in molecular weight and glycosylation pattern than the native molecules, depending upon the expression system. Expression of CRF₂ R nucleic acids in bacteria such as E. coli provides non-glycosylated molecules. Functional mutant analogs of mammalian CRF₂ R having inactivated N-glycosylation sites may be produced by oligonucleotide synthesis and ligation or by site-specific mutagenesis techniques. These analog proteins may be produced in a homogeneous, reduced-carbohydrate form in good yield using yeast expression systems. N-glycosylation sites in eukaryotic proteins are generally characterized by the amino acid triplet Asn-A₁ -Z, where A₁ is any amino acid except Pro, and Z is Ser or Thr. In this sequence, asparagine provides a side chain amino group for covalent attachment of carbohydrate. Such a site can be eliminated by substituting another amino acid for Asn or for residue Z, deleting Asn or Z, or inserting a non-Z amino acid between A₁ and Z, or an amino acid other than Asn between Asn and A₁.

Proteins which are biologically active and substantially similar to CRF₂ R proteins may also be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for biological activity. For example, cysteine residues can be deleted or replaced with other amino acids to prevent formation of incorrect intramolecular disulfide bridges upon renaturation. Other approaches to mutagenesis involve modification of adjacent dibasic amino acid residues to enhance expression in yeast systems in which KEX2 protease activity is present. Generally, substitutions should be made conservatively, i.e., the most preferred substitute amino acids are those having physicochemical characteristics resembling those of the residue to be replaced. When a substitution, deletion, or insertion strategy is adopted, the potential effect of the deletion or insertion on biological activity should be considered utilizing, for example, a binding assay such as that disclosed within the Examples.

Mutations in nucleotide sequences constructed for expression of proteins which are substantially similar to CRF₂ receptors should preserve the reading frame phase of the coding sequences. Furthermore, the mutations should preferably not create complementary regions that could hybridize to produce secondary mRNA structures, such as loops or hairpins, which would adversely affect translation of the receptor mRNA. Although a mutation site may be predetermined, it is not necessary that the nature of the mutation per se be predetermined. For example, in order to select for optimum characteristics of mutants at a given site, random mutagenesis may be conducted at the target codon, and the expressed CRF₂ R mutants screened for the biological activity.

Not all mutations in the nucleotide sequence which encodes CRF₂ R will be expressed in the final product. For example, nucleotide substitutions may be made to enhance expression primarily to avoid secondary structure loops in the transcribed mRNA, or to provide codons that are more readily translated by the selected host, e.g., the well-known, E. coli preference codons for E. coli expression.

Mutations may be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific mutagenesis procedures may be employed to provide an altered gene having particular codons altered according to the substitution, deletion, or insertion required. Exemplary methods of making the alterations set forth above are disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik, Bio Techniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); Sambrook et al. (Molecular cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, 1989); and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are incorporated by reference herein.

CRF₂ receptors, as well as substantially similar derivatives or analogs may be used as therapeutic reagents, immunogens, reagents in receptor-based immunoassays, or as binding agents for affinity purification procedures of CRF, sauvagine, urotensin I, other related molecules, or other binding ligands such as anti-CRF₂ receptor antibodies. Moreover, CRF₂ receptors of the present invention may be utilized to screen compounds for CRF₂ receptors agonist or antagonistic activity. CRF₂ receptor proteins may also be covalently bound through reactive side groups to various insoluble substrates, such as cyanogen bromine-activated, bisoxirane-activated, carbonyldiimidazole-activated, or tosyl-activated, agarose structures, or by adsorbing to polyolefin surfaces (with or without glutaraldehyde cross-linking). Once bound to a substrate, CRF₂ R may be used to selectively bind (for purposes of assay or purification) anti-CRF₂ R antibodies or CRF.

ISOLATION OF CRF₂ RECEPTOR cDNA CLONES

As noted above, the present invention provides isolated nucleic acid molecules which encode CRF₂ receptors. Briefly, nucleic acid molecules which encode CRF₂ receptors of the present invention may be readily isolated from a variety of warm-blooded animals, including for example, humans, macaques, horses, cattle, sheep, pigs, dogs, cats, rats and mice. Particularly preferred tissues from which nucleic acid molecules which encode CRF₂ receptors may be isolated include brain and neural tissues such as the hypothalamus, hippocampus, and frontal cortex, as well as other tissues such as the lung, heart, skeletal muscle, or kidney. Nucleic acid molecules which encode CRF₂ receptors of the present invention may be readily isolated from conventionally prepared cDNA libraries (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, NY, 1989) or from commercially obtained libraries (e.g., Stratagene, LaJolla, Calif.) utilizing the disclosure provided herein. Particularly preferred methods for obtaining isolated nucleic acid molecules which encode CRF₂ receptors of the present invention are described in more detail below in Example 1 (see also Sequence I.D. Nos. 1 and 3).

As noted above, within particularly preferred embodiments of the invention, isolated nucleic acid molecules are provided which encode human CRF₂ receptors. Briefly, such nucleic acid molecules may be readily obtained by probing a human cDNA library either with a specific sequence as described below in Example 1, or with a rat sequence (e.g., Sequence I.D. Nos. 1 or 3) under conditions of low stringency (e.g., 35% formamide, 5×SSC, 5×Denharts, 0.1% SDS, 100 ug/ml salmon sperm nucleic acid, at 42° C. for 12 hours. This may be followed by extensive washing with 2×SSC containing 0.2% SDS at 50° C. Suitable cDNA libraries may be obtained from commercial sources (e.g., Stratagene, LaJolla, Calif.), or prepared utilizing standard techniques (see, e.g. Sambrook et al., supra).

PRODUCTION OF RECOMBINANT CRF₂ RECEPTORS

As noted above, the present invention also provides recombinant expression vectors which include synthetic or cDNA-derived nucleic acid fragments encoding CRF₂ receptors or substantially similar proteins, which are operably linked to suitable transcriptional or translation regulatory elements derived from mammalian, microbial, viral or insect genes. Such regulatory elements include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and, within preferred embodiments, sequences which control the termination of transcription and translation. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants may additionally be incorporated. Nucleic acid regions are operably linked when they are functionally related to each other. For example, nucleic acid for a signal peptide (secretory leader) is operably linked to nucleic acid for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. Generally, operably linked means contiguous and, in the case of secretory leaders, contiguous and in reading frame.

Expression vectors may also contain nucleic acid sequences necessary to direct the secretion of a polypeptide of interest. Such nucleic acid sequences may include at least one secretory signal sequence. Representative secretory signals include the alpha factor signal sequence (pre-pro sequence; Kurjan and Herskowitz, Cell 30:933-943, 1982; Kurjan et al., U.S. Pat. No. 4,546,082; Brake, EP 116,201), the PHO5 signal sequence (Beck et al., WO 86/00637), the BAR1 secretory signal sequence (MacKay et al., U.S. Pat. No. 4,613,572; MacKay, WO 87/002670), the SUC2 signal sequence (Carlson et al., Mol. Cell. Biol. 3:439-447, 1983), the α-1-antitrypsin signal sequence (Kurachi et al., Proc. Natl. Acad. Sci. USA 78:6826-6830, 1981), the β-2 plasmin inhibitor signal sequence (Tone et al., J. Biochem. (Tokyo) 102:1033-1042, 1987), the tissue plasminogen activator signal sequence (Pennica et al., Nature 301:214-221, 1983), the E. coli PhoA signal sequence (Yuan et al., J. Biol. Chem. 265:13528-13552, 1990) or any of the bacterial signal sequences reviewed, for example, by Oliver (Ann. Rev. Microbiol. 39:615-649, 1985). Alternatively, a secretory signal sequence may be synthesized according to the rules established, for example, by von Heinje (Eur. J. Biochem. 133:17-21, 1983; J. Mol. Biol. 184:99-105, 1985; Nuc. Acids Res. 14:4683-4690, 1986).

For expression, a nucleic acid molecule encoding a CRF₂ receptor is inserted into a suitable expression vector, which in turn is used to transform or transfect appropriate host cells for expression. Host cells for use in practicing the present invention include mammalian, avian, plant, insect, bacterial and fungal cells. Preferred eukaryotic cells include cultured mammalian cell lines (e.g., rodent or human cell lines) and fungal cells, including species of yeast (e.g., Saccharomyces spp., particularly S. cerevisiae, Schizosaccharomyces spp., or Kluyveromyces spp.) or filamentous fungi (e.g., Aspergillus spp., Neurospora spp.). Strains of the yeast Saccharomyces cerevisiae are particularly preferred. Methods for producing recombinant proteins in a variety of prokaryotic and eukaryotic host cells are generally known in the art (see "Gene Expression Technology," Methods in Enzymology, Vol. 185, Goeddel (ed.), Academic Press, San Diego, Calif, 1990; see also, "Guide to Yeast Genetics and Molecular Biology," Methods in Enzymology, Guthrie and Fink (eds.) Academic Press, San Diego, Calif, 1991). In general, a host cell will be selected on the basis of its ability to produce the protein of interest at a high level or its ability to carry out at least some of the processing steps necessary for the biological activity of the protein. In this way, the number of cloned nucleic acid sequences which must be transfected into the host cell may be minimized and overall yield of biologically active protein may be maximized.

Suitable yeast vectors for use in the present invention include YRp7 (Struhl et al., Proc. Natl. Acad. Sci. USA 76:1035-1039, 1978), YEp13 (Broach et al., Gene 8:121-133, 1979), POT vectors (Kawasaki et al., U.S. Pat. No. 4,931,373, which is incorporated by reference herein), pJDB249 and pJDB219 (Beggs, Nature 275:104-108, 1978) and derivatives thereof. Such vectors will generally include a selectable marker, which may be one of any number of genes that exhibit a dominant phenotype for which a phenotypic assay exists to enable transformants to be selected. Preferred selectable markers are those that complement host cell auxotrophy, provide antibiotic resistance or enable a cell to utilize specific carbon sources, and include LEU2 (Broach et al., ibid.), URA3 (Botstein et al., Gene 8:17, 1979), HIS3 (Struhl et al., ibid.) or POT1 (Kawasaki et al., ibid.). Another suitable selectable marker is the CAT gene, which confers chloramphenicol resistance on yeast cells.

Preferred promoters for use in yeast include promoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem. 255:12073-12080, 1980; Alber and Kawasaki, J. Mol. Appl. Genet. 1:419-434, 1982; Kawasaki, U.S. Pat. No. 4,599,311) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering of Microorganisms for Chemicals, Hollaender et al. (eds.), p. 355, Plenum, New York, 1982; Ammerer, Meth. Enzymol. 101:192-201, 1983). In this regard, particularly preferred promoters are the TPI1 promoter (Kawasaki, U.S. Pat. No. 4,599,311, 1986) and the ADH2-4^(c) promoter (Russell et al., Nature 304:652-654, 1983; Irani and Kilgore, U.S. patent application Ser. No. 07/784,653, which is incorporated herein by reference). The expression units may also include a transcriptional terminator, such as the TPI1 terminator (Alber and Kawasaki, ibid.).

In addition to yeast, proteins of the present invention can be expressed in filamentous fungi, for example, strains of the fungi Aspergillus (McKnight et al., U.S. Pat. No. 4,935,349, which is incorporated herein by reference). Examples of useful promoters include those derived from Aspergillus nidulans glycolytic genes, such as the ADH3 promoter (McKnight et al., EMBO J. 4:2093-2099, 1985) and the tpiA promoter. An example of a suitable terminator is the ADH3 terminator (McKnight et al., ibid., 1985). The expression units utilizing such components are cloned into vectors that are capable of insertion into the chromosomal nucleic acid of Aspergillus.

Techniques for transforming fungi are well known in the literature, and have been described, for instance, by Beggs (ibid.), Hinnen et al. (Proc. Natl. Acad. Sci. USA 75:1929-1933, 1978), Yelton et al. (Proc. Natl. Acad. Sci. USA 81:1740-1747, 1984), and Russell (Nature 301:167-169, 1983). The genotype of the host cell will generally contain a genetic defect that is complemented by the selectable marker present on the expression vector. Choice of a particular host and selectable marker is well within the level of ordinary skill in the art. To optimize production of the heterologous proteins in yeast, for example, it is preferred that the host strain carries a mutation, such as the yeast pep4 mutation (Jones, Genetics 85:23-33, 1977), which results in reduced proteolytic activity.

In addition to fungal cells, cultured mammalian cells may be used as host cells within the present invention. Preferred cultured mammalian cells for use in the present invention include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), and 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) cell lines. A preferred BHK cell line is the BHK 570 cell line (deposited with the American Type Culture Collection under accession number CRL 10314). In addition, a number of other mammalian cell lines may be used within the present invention, including Rat Hep I (ATCC No. CRL 1600), Rat Hep II (ATCC No. CRL 1548), TCMK (ATCC No. CCL 139), Human lung (ATCC No. CCL 75.1), Human hepatoma (ATCC No. HTB-52), Hep G2 (ATCC No. HB 8065), Mouse liver (ATCC No. CCL 29.1), NCTC 1469 (ATCC No. CCL 9.1), SP2/0-Ag14 (ATCC No. 1581), HIT-T15 (ATCC No. CRL 1777), and RINm 5AHT₂ B (Orskov and Nielson, FEBS 229(1):175-178, 1988).

Mammalian expression vectors for use in carrying out the present invention should include a promoter capable of directing the transcription of a cloned gene or cDNA. Preferred promoters include viral promoters and cellular promoters. Viral promoters include the immediate early cytomegalovirus promoter (Boshart et al., Cell 41:521-530, 1985) and the SV40 promoter (Subramani et al., Mol. Cell. Biol. 1:854-864, 1981). Cellular promoters include the mouse metallothionein-1 promoter (Palmiter et al., U.S. Pat. No. 4,579,821), a mouse V_(j) promoter (Bergman et al., Proc. Natl. Acad. Sci. USA 81:7041-7045, 1983; Grant et al., Nuc. Acids Res. 15:5496, 1987) and a mouse V_(H) promoter (Loh et al., Cell 33:85-93, 1983). A particularly preferred promoter is the major late promoter from Adenovirus 2 (Kaufman and Sharp, Mol. Cell. Biol. 2:1304-13199, 1982). Such expression vectors may also contain a set of RNA splice sites located downstream from the promoter and upstream from the nucleic acid sequence encoding the peptide or protein of interest. Preferred RNA splice sites may be obtained from SV40, adenovirus and/or immunoglobulin genes. Alternatively, within certain embodiments RNA splice sites may be located downstream from the nucleic acid sequence encoding the peptide or protein of interest. Also contained in the expression vectors is a polyadenylation signal located downstream of the coding sequence of interest. Suitable polyadenylation signals include the early or late polyadenylation signals from SV40 (Kaufman and Sharp, ibid.), the polyadenylation signal from the Adenovirus 5 E1B region and the human growth hormone gene terminator (DeNoto et al., Nuc. Acids Res. 9:3719-3730, 1981). The expression vectors may include a noncoding viral leader sequence, such as the Adenovirus 2 tripartite leader, located between the promoter and the RNA splice sites. Preferred vectors may also include enhancer sequences, such as the SV40 enhancer and the mouse 1 enhancer (Gillies, Cell 33:717-728, 1983). Expression vectors may also include sequences encoding the adenovirus VA RNAs. Suitable vectors can be obtained from commercial sources (e.g., Invitrogen, San Diego, Calif.; Stratagene, La Jolla, Calif.).

Cloned nucleic acid sequences may be introduced into cultured mammalian cells by, for example, calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J. 1:841-845, 1982), or DEAE-dextran mediated transfection (Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987), which are incorporated herein by reference. To identify cells that have stably integrated the cloned nucleic acid, a selectable marker is generally introduced into the cells along with the gene or cDNA of interest. Preferred selectable markers for use in cultured mammalian cells include genes that confer resistance to drugs, such as neomycin, hygromycin, and methotrexate. The selectable marker may be an amplifiable selectable marker. Preferred amplifiable selectable markers are the DHFR gene and the neomycin resistance gene. Selectable markers are reviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass., which is incorporated herein by reference). The choice of selectable markers is well within the level of ordinary skill in the art.

Selectable markers may be introduced into the cell on a separate vector at the same time as the CRF₂ receptor sequence, or they may be introduced on the same vector. If on the same vector, the selectable marker and the CRF₂ receptor sequence may be under the control of different promoters or the same promoter, the latter arrangement producing a dicistronic message. Constructs of this type are known in the art (for example, Levinson and Simonsen, U.S. Pat. No. 4,713,339). It may also be advantageous to add additional nucleic acid, known as "carrier nucleic acid" to the mixture which is introduced into the cells.

Transfected mammalian cells are allowed to grow for a period of time, typically 1-2 days, to begin expressing the nucleic acid sequence(s) of interest. Drug selection is then applied to select for growth of cells that are expressing the selectable marker in a stable fashion. For cells that have been transfected with an amplifiable selectable marker the drug concentration may be increased in a stepwise manner to select for increased copy number of the cloned sequences, thereby increasing expression levels. Cells expressing the introduced sequences are selected and screened for production of the protein of interest in the desired form or at the desired level. Cells which satisfy these criteria may then be cloned and scaled up for production.

Preferred prokaryotic host cells for use in carrying out the present invention are strains of the bacteria Escherichia coli, although Bacillus and other genera are also useful. Techniques for transforming these hosts and expressing foreign nucleic acid sequences cloned therein are well known in the art (see, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1982; or Sambrook et al., supra). Vectors used for expressing cloned nucleic acid sequences in bacterial hosts will generally contain a selectable marker, such as a gene for antibiotic resistance, and a promoter that functions in the host cell. Appropriate promoters include the trp (Nichols and Yanofsky, Meth. Enzymol. 101:155-164, 1983), lac (Casadaban et al., J. Bacteriol. 143:971-980, 1980), and phage k (Queen, J. Mol. Appl. Genet. 2:1-10, 1983) promoter systems. Plasmids useful for transforming bacteria include pBR322 (Bolivar et al., Gene 2:95-113, 1977), the pUC plasmids (Messing, Meth. Enzymol. 101:20-78, 1983; Vieira and Messing, Gene 19:259-268, 1982), pCQV2 (Queen, ibid.), pMAL-2 (New England Biolabs, Beverly, Mass.) and derivatives thereof. Plasmids may contain both viral and bacterial elements.

Given the teachings provided herein, promoters, terminators and methods for introducing expression vectors encoding CRF₂ receptors of the present invention into plant, avian and insect cells would be evident to those of skill in the art. The use of baculoviruses, for example, as vectors for expressing heterologous nucleic acid sequences in insect cells has been reviewed by Atkinson et al. (Pestic. Sci. 28:215-224,1990). In addition, the use of Agrobacterium rhizogenes as vectors for expressing genes in plant cells has been reviewed by Sinkar et al. (J. Biosci. (Bangalore) 11:47-58, 1987).

Host cells containing nucleic acid molecules of the present invention are then cultured to express a nucleic acid molecule encoding a CRF₂ receptor. The cells are cultured according to standard methods in a culture medium containing nutrients required for growth of the chosen host cells. A variety of suitable media are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals, as well as other components, e.g., growth factors or serum, that may be required by the particular host cells. The growth medium will generally select for cells containing the nucleic acid molecules by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker on the nucleic acid construct or co-transfected with the nucleic acid construct.

Suitable growth conditions for yeast cells, for example, include culturing in a chemically defined medium, comprising a nitrogen source, which may be a non-amino acid nitrogen source or a yeast extract, inorganic salts, vitamins and essential amino acid supplements at a temperature between 4° C. and 37° C., with 30° C. being particularly preferred. The pH of the medium is preferably maintained at a pH greater than 2 and less than 8, more preferably pH 5-6. Methods for maintaining a stable pH include buffering and constant pH control. Preferred agents for pH control include sodium hydroxide. Preferred buffering agents include succinic acid and Bis-Tris (Sigma Chemical Co., St. Louis, Mo.). Due to the tendency of yeast host cells to hyperglycosylate heterologous proteins, it may be preferable to express the CRF₂ receptors of the present invention in yeast cells having a defect in a gene required for asparagine-linked glycosylation. Such cells are preferably grown in a medium containing an osmotic stabilizer. A preferred osmotic stabilizer is sorbitol supplemented into the medium at a concentration between 0.1M and 1.5M, preferably at 0.5M or 1.0M. Cultured mammalian cells are generally cultured in commercially available serum-containing or serum-free media. Selection of a medium and growth conditions appropriate for the particular cell line used is within the level of ordinary skill in the art.

CRF₂ receptors may also be expressed in non-human transgenic animals, particularly transgenic warm-blooded animals. Methods for producing transgenic animals, including mice, rats, rabbits, sheep and pigs, are known in the art and are disclosed, for example, by Hammer et al. (Nature 315:680-683, 1985), Palmiter et al. (Science 222:809-814, 1983), Brinster et al. (Proc. Natl. Acad. Sci. USA 82:4438-4442, 1985), Palmiter and Brinster (Cell 41:343-345, 1985) and U.S. Pat. No. 4,736,866, which are incorporated herein by reference. Briefly, an expression unit, including a nucleic acid sequence to be expressed together with appropriately positioned expression control sequences, is introduced into pronuclei of fertilized eggs. Introduction of nucleic acid is commonly done by microinjection. Integration of the injected nucleic acid is detected by blot analysis of nucleic acid from tissue samples, typically samples of tail tissue. It is generally preferred that the introduced nucleic acid be incorporated into the germ line of the animal so that it is passed on to the animal's progeny.

Within particularly preferred embodiments of the invention, "knockout" animals may be developed from embryonic stem cells through the use of homologous recombination (Capecchi, Science 244:1288-1292, 1989) or antisense oligonucleotide (Stein and Chen, Science 261(5124):1004-1012, 1993; Milligan et al., Semin. Conc. Biol. 3(6):391-398, 1992).

Within a preferred embodiment of the invention, a transgenic animal, such as a mouse, is developed by targeting a mutation to disrupt a CRF₂ receptor sequence (see Mansour et al., "Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: A general strategy for targeting mutations to non-selectable genes," Nature 336:348-352, 1988). Such animals may readily be utilized as a model to study the role of the CRF₂ receptor in metabolism.

CRF₂ RECEPTOR PEPTIDES

As noted above, the present invention also provides CRF₂ receptor peptides. Within the context of the present invention, CRF₂ receptor peptides should be understood to include portions of a CRF₂ receptor or derivatives thereof discussed above, which do not contain transmembrane domains, and which are at least 8, and more preferably 10 or greater amino acids in length. Briefly, the structure of the CRF₂ receptor as well as putative transmembrane domains may be predicted from the primary translation products using the hydrophopicity plot function of, for example, P/C Gene or Intelligenetics Suite (Intelligenetics, Mt. View, Calif.), or according to the methods described by Kyte and Doolittle (J. Mol. Biol. 157:105-132, 1982). While not wishing to be bound by a graphical representation, based upon this hydrophopicity analysis, CRF₂ receptors are believed to have the general structure shown in FIGS. 1 and 2. In particular, these receptors are believed to comprise an extracellular amino-terminal domain, three extracellular loop domains and four intracellular loop domains each separated by a transmembrane domain.

Within one aspect of the invention, isolated CRF₂ receptor peptides are provided comprising the extracellular amino-terminal domain of a CRF₂ receptor. Within a preferred embodiment, an isolated CRF₂ receptor peptide is provided comprising the sequence of amino acids shown in Sequence I.D. No. 4, from amino acid 1 to amino acid number 118. Within another embodiment, an isolated CRF2 receptor peptide is provided comprising the sequence of amino acids shown in Sequence ID No. 2 from amino acid number 1 to amino acid number 138.

CRF₂ receptor peptides may be prepared by, among other methods, culturing suitable host/vector systems to produce the recombinant translation products of the present invention. Supernatants from such cell lines may then be treated by a variety of purification procedures in order to isolate the CRF₂ receptor peptide. For example, the supernatant may be first concentrated using commercially available protein concentration filters, such as an Amicon or Millipore Pellicon ultrafiltration unit. Following concentration, the concentrate may be applied to a suitable purification matrix such as, for example, CRF or an anti-CRF₂ receptor antibody bound to a suitable support. Alternatively, anion or cation exchange resins may be employed in order to purify the receptor or peptide. Finally, one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps may be employed to further purify the CRF₂ receptor peptide.

Alternatively, CRF₂ receptor peptides may also be prepared utilizing standard polypeptide synthesis protocols, and purified utilizing the above-described procedures.

A CRF₂ receptor peptide is deemed to be "isolated" or purified within the context of the present invention, if only a single band is detected subsequent to SDS-polyacrylamide gel analysis followed by staining with Coomassie Brilliant Blue.

ANTIBODIES To CRF₂ RECEPTORS

Within one aspect of the present invention, CRF₂ receptors, including derivatives thereof, as well as portions or fragments of these proteins such as the CRF₂ receptor peptides discussed above, may be utilized to prepare antibodies which specifically bind to CRF₂ receptors. Within the context of the present invention the term "antibodies" includes polyclonal antibodies, monoclonal antibodies, fragments thereof such as F(ab')₂ and Fab fragments, as well as recombinantly produced binding partners. These binding partners incorporate the variable regions from a gene which encodes a specifically binding monoclonal antibody. Antibodies are defined to be specifically binding if they bind to the CRF₂ receptor with a K_(a) of greater than or equal to 10⁷ M⁻¹. The affinity of a monoclonal antibody or binding partner may be readily determined by one of ordinary skill in the art (see Scatchard, Ann. N.Y. Acad. Sci. 51:660-672, 1949).

Polyclonal antibodies may be readily generated by one of ordinary skill in the art from a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, or rats. Briefly, the CRF₂ receptor is utilized to immunize the animal through intraperitoneal, intramuscular, intraocular, or subcutaneous injections. The immunogenicity of a CRF₂ receptor or CRF₂ receptor peptide may be increased through the use of an adjuvant such as Freund's complete or incomplete adjuvant. Following several booster immunizations, small samples of serum are collected and tested for reactivity to the CRF₂ receptor. A variety of assays may be utilized in order to detect antibodies which specifically bind to a CRF₂ receptor. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: Countercurrent Immuno-Electrophoresis (CIEP), Radioimmunoassays, Radioimmunoprecipitations, Enzyme-Linked Immuno-Sorbent Assays (ELISA), Dot Blot assays, Inhibition or Competition assays, and sandwich assays (see U.S. Pat. Nos. 4,376,110 and 4,486,530; see also Antibodies: A Laboratory Manual, supra). Particularly preferred polyclonal antisera will give a signal that is at least three times greater than background. Once the titer of the animal has reached a plateau in terms of its reactivity to the CRF₂ receptor, larger quantities of polyclonal antisera may be readily obtained either by weekly bleedings, or by exsanguinating the animal.

Monoclonal antibodies may also be readily generated using well-known techniques (see U.S. Pat. Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993; see also Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988). Briefly, within one embodiment a subject animal such as a rat or mouse is injected with a form of CRF₂ receptor suitable for generating an immune response against the CRF₂ receptor. Representative examples of suitable forms include, among others, cells which express the CRF₂ receptor, or peptides which are based upon the CRF₂ receptor sequence. Additionally, many techniques are known in the art for increasing the resultant immune response, for example, by coupling the receptor or receptor peptides to another protein such as ovalbumin or keyhole limpet hemocyanin (KLH), or through the use of adjuvants such as Freund's complete or incomplete adjuvant. The initial immunization may be through intraperitoneal, intramuscular, intraocular, or subcutaneous routes.

Between one and three weeks after the initial immunization the animal may be reimmunized with another booster immunization. The animal may then be test bled and the serum tested for binding to the CRF₂ receptor using assays as described above. Additional immunizations may also be accomplished until the animal has plateaued in its reactivity to the CRF₂ receptor. The animal may then be given a final boost of CRF₂ receptor or CRF₂ receptor peptide, and three to four days later sacrificed. At this time, the spleen and lymph nodes may be harvested and disrupted into a single cell suspension by passing the organs through a mesh screen or by rupturing the spleen or lymph node membranes which encapsidate the cells. Within one embodiment the red cells are subsequently lysed by the addition of a hypotonic solution, followed by immediate return to isotonicity.

Within another embodiment, suitable cells for preparing monoclonal antibodies are obtained through the use of in vitro immunization techniques. Briefly, an animal is sacrificed, and the spleen and lymph node cells are removed as described above. A single cell suspension is prepared, and the cells are placed into a culture containing a form of the CRF₂ receptor that is suitable for generating an immune response as described above. Subsequently, the lymphocytes are harvested and fused as described below.

Cells which are obtained through the use of in vitro immunization or from an immunized animal as described above may be immortalized by transfection with a virus such as the Epstein-Barr virus (EBV) (see Glasky and Reading, Hybridoma 8(4):377-389, 1989). Alternatively, within a preferred embodiment, the harvested spleen and/or lymph node cell suspensions are fused with a suitable myeloma cell in order to create a "hybridoma" which secretes monoclonal antibodies. Suitable myeloma lines are preferably defective in the construction or expression of antibodies, and are additionally syngeneic with the cells from the immunized animal. Many such myeloma cell lines are well known in the art and may be obtained from sources such as the American Type Culture Collection (ATCC), Rockville, Md. (see Catalogue of Cell Lines & Hybridomas, 6th ed., ATCC, 1988). Representative myeloma lines include: for humans, UC 729-6 (ATCC No. CRL 8061), MC/CAR-Z2 (ATCC No. CRL 8147), and SKO-007 (ATCC No. CRL 8033); for mice, SP2/0-Ag14 (ATCC No. CRL 1581), and P3X63Ag8 (ATCC No. TIB 9); and for rats, Y3-Ag1.2.3 (ATCC No. CRL 1631), and YB2/0 (ATCC No. CRL 1662). Particularly preferred fusion lines include NS-1 (ATCC No. TIB 18) and P3X63-Ag 8.653 (ATCC No. CRL 1580), which may be utilized for fusions with either mouse, rat, or human cell lines. Fusion between the myeloma cell line and the cells from the immunized animal may be accomplished by a variety of methods, including the use of polyethylene glycol (PEG) (see Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988) or electrofusion (see Zimmerman and Vienken, J. Membrane Biol. 67:165-182, 1982).

Following the fusion, the cells are placed into culture plates containing a suitable medium, such as RPMI 1640 or DMEM (Dulbecco's Modified Eagles Medium) (JRH Biosciences, Lenexa, Kans.). The medium may also contain additional ingredients, such as Fetal Bovine Serum ("FBS," i.e., from Hyclone, Logan, Utah, or JRH Biosciences), thymocytes which were harvested from a baby animal of the same species as was used for immunization, or agar to solidify the medium. Additionally, the medium should contain a reagent which selectively allows for the growth of fused spleen and myeloma cells. Particularly preferred is the use of HAT (hypoxanthine, aminopterin, and thymidine) (Sigma Chemical Co., St. Louis, Mo.). After about seven days, the resulting fused cells or hybridomas may be screened in order to determine the presence of antibodies which recognize the CRF₂ receptor. Following several clonal dilutions and reassays, a hybridoma producing antibodies which bind to CRF₂ receptor may be isolated.

Other techniques may also be utilized to construct monoclonal antibodies (see Huse et al., "Generation of a Large Combinational Library of the Immunoglobulin Repertoire in Phage Lambda," Science 246:1275-1281, December 1989; see also Sastry et al., "Cloning of the Immunological Repertoire in Escherichia coli for Generation of Monoclonal Catalytic Antibodies: Construction of a Heavy Chain Variable Region-Specific cDNA Library," Proc. Natl. Acad. Sci. USA 86:5728-5732, August 1989; see also Alting-Mees et al., "Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas," Strategies in Molecular Biology 3:1-9, January 1990; these references describe a commercial system available from Stratacyte, La Jolla, Calif., which enables the production of antibodies through recombinant techniques). Briefly, mRNA is isolated from a B cell population and utilized to create heavy and light chain immunoglobulin cDNA expression libraries in the kIMMUNOZAP(H) and kIMMUNOZAP(L) vectors. These vectors may be screened individually or co-expressed to form Fab fragments or antibodies (see Huse et al., supra; see also Sastry et al., supra). Positive plaques may subsequently be converted to a non-lytic plasmid which allows high level expression of monoclonal antibody fragments from E. coli.

Similarly, binding partners may also be constructed utilizing recombinant nucleic acid techniques to incorporate the variable regions of a gene which encodes a specifically binding antibody. The construction of these proteins may be readily accomplished by one of ordinary skill in the art (see Larrick et al., "Polymerase Chain Reaction Using Mixed Primers: Cloning of Human Monoclonal Antibody Variable Region Genes From Single Hybridoma Cells," Biotechnology 7:934-938, September 1989; Riechmann et al., "Reshaping Human Antibodies for Therapy," Nature 332:323-327, 1988; Roberts et al., "Generation of an Antibody with Enhanced Affinity and Specificity for its Antigen by Protein Engineering," Nature 328:731-734, 1987; Verhoeyen et al., "Reshaping Human Antibodies: Grafting an Antilysozyme Activity," Science 239:1534-1536, 1988; Chaudhary et al., "A Recombinant Immunotoxin Consisting of Two Antibody Variable Domains Fused to Pseudomonas Exotoxin," Nature 339:394-397, 1989; see also, U.S. Pat. No. 5,132,405 entitled "Biosynthetic Antibody Binding Sites"), given the disclosure provided herein. Briefly, within one embodiment, nucleic acid molecules encoding CRF₂ receptor-specific antigen binding domains are amplified from hybridomas which produce a specifically binding monoclonal antibody, and inserted directly into the genome of a cell which produces human antibodies (see Verhoeyen et al., supra; see also Reichmann et al., supra). This technique allows the antigen-binding site of a specifically binding mouse or rat monoclonal antibody to be transferred into a human antibody. Such antibodies are preferable for therapeutic use in humans because they are not as antigenic as rat or mouse antibodies.

Alternatively, the antigen-binding sites (variable region) may be either linked to, or inserted into, another completely different protein (see Chaudhary et al., supra), resulting in a new protein with antigen-binding sites of the antibody as well as the functional activity of the completely different protein. As one of ordinary skill in the art will recognize, the antigen-binding sites or CRF₂ receptor binding domain of the antibody may be found in the variable region of the antibody. Furthermore, nucleic acid sequences which encode smaller portions of the antibody or variable regions which specifically bind to mammalian CRF₂ receptor may also be utilized within the context of the present invention. These portions may be readily tested for binding specificity to the CRF₂ receptor utilizing assays described below.

Within a preferred embodiment, genes which encode the variable region from a hybridoma producing a monoclonal antibody of interest are amplified using oligonucleotide primers for the variable region. These primers may be synthesized by one of ordinary skill in the art, or may be purchased from commercially available sources. Stratacyte (La Jolla, Calif.) sells primers for mouse and human variable regions including, among others, primers for V_(Ha), V_(Hb), V_(Hc), V_(Hd), C_(Hl), V_(L) and C_(L) regions. These primers may be utilized to amplify heavy or light chain variable regions, which may then be inserted into vectors such as IMMUNOZAP*(H) or IMMUNOZAP*(L) (Stratacyte), respectively. These vectors may then be introduced into E. coli for expression. Utilizing these techniques, large amounts of a single-chain protein containing a fusion of the V_(H) and V_(L) domains may be produced (see Bird et al., Science 242:423-426, 1988).

Other "antibodies" which may also be prepared utilizing the disclosure provided herein, and thus which are also deemed to fall within the scope of the present invention include humanized antibodies (e.g., U.S. Pat. No. 4,816,567 and WO 94/10332), micobodies (e.g., WO 94/09817) and transgenic antibodies (e.g., GB 2 272 440).

Once suitable antibodies have been obtained, they may be isolated or purified by many techniques well known to those of ordinary skill in the art (see Antibodies: A Laboratory Manual, supra). Suitable techniques include peptide or protein affinity columns, HPLC or RP-HPLC, purification on protein A or protein G columns, or any combination of these techniques. Within the context of the present invention, the term "isolated" as used to define antibodies or binding partners means "substantially free of other blood components."

Antibodies of the present invention have many uses. For example, antibodies may be utilized in flow cytometry to sort CRF₂ receptor-bearing cells, or to histochemically stain CRF₂ receptor-bearing, tissues. Briefly, in order to detect CRF₂ receptors on cells, the cells (or tissue) are incubated with a labeled antibody which specifically binds to CRF₂ receptors, followed by detection of the presence of bound antibody. These steps may also be accomplished with additional steps such as washings to remove unbound antibody. Representative examples of suitable labels, as well as methods for conjugating or coupling antibodies to such labels are described in more detail below.

In addition, purified antibodies may also be utilized therapeutically to block the binding of CRF or other CRF₂ receptor substrates such as sauvagine or urotensin I to the CRF₂ receptor in vitro or in vivo. Briefly, blocking antibodies are those antibodies that bind to CRF₂ receptor epitopes in such a way as to prevent CRF from binding to the receptor, or to prevent CRF from effecting signal transduction. As noted above, a variety of assays may be utilized to detect antibodies which block or inhibit the binding of CRF to the CRF₂ receptor, including inter alia, inhibition and competition assays noted above. Within one embodiment, monoclonal antibodies (prepared as described above) are assayed for binding to the CRF₂ receptor in the absence of CRF, as well as in the presence of varying concentrations of CRF. Blocking antibodies are identified as those which, for example, bind to CRF₂ receptors and, in the presence of CRF, block or inhibit the binding of CRF to the CRF₂ receptor.

Antibodies of the present invention may also be coupled or conjugated to a variety of other compounds for either diagnostic or therapeutic use. Such compounds include, for example, toxic molecules, molecules which are nontoxic but which become toxic upon exposure to a second compound, and radionuclides. Representative examples of such molecules are described in more detail below.

Antibodies which are to be utilized therapeutically are preferably provided in a therapeutic composition comprising the antibody or binding partner and a physiologically acceptable carrier or diluent. Suitable carriers or diluents include, among others, neutral buffered saline or saline, and may also include additional excipients or stabilizers such as buffers, sugars such as glucose, sucrose, or dextrose, chelating agents such as EDTA, and various preservatives.

ISOLATION AND USE OF COMPOUNDS WHICH BIND THE CRF₂ RECEPTOR, INCLUDING AGONISTS AND ANTAGONISTS

As noted above, the present invention provides a variety of methods for detecting the presence of compounds which bind to CRF₂ receptors. For example, within one embodiment of the invention methods for detecting such compounds are provided, comprising the steps of (a) exposing one or more compounds to cells that express CRF₂ receptors under conditions and for a time sufficient to allow binding of the compounds to the receptors, and (b) isolating compounds which bind to the receptors, such that the presence of a compound which binds to a CRF₂ receptor may be detected. As utilized herein, conditions sufficient to allow binding of compounds to cells that express CRF₂ receptors generally range from pH 6.8 to 7.5, and include a suitable buffer such as Tris-HCl or PBS, either with or without bovine serum albumin ("BSA"). Such buffers may also include magnesium at a concentration of 5 to 20 mM, as well as protease inhibitors such as bacitracin or aprotinin. Sufficient time for the binding of compounds to cells that express CRF₂ receptors generally ranges from between 30 and 150 minutes after exposure.

Within other aspects of the present invention, methods for detecting the presence of a compound which binds to CRF₂ receptors are provided, comprising the steps of (a) exposing one or more compounds to a CRF₂ receptor N-terminal extracellular domain under conditions and for a time sufficient to allow binding of a compound to the N-terminal extracellular domain, and (b) isolating compounds which bind to the CRF₂ receptor N-terminal extracellular domain, such that the presence of a compound which binds to a CRF₂ receptor may be detected. Such assays may take a variety of forms, including for example, as Enzyme Linked ImmnoSorbent Assay, "Sandwich" assay, or the like. Within one embodiment, the compounds are labeled with an agent selected from the group consisting of fluorescent molecules, enzymes, and radionuclides.

In addition to providing assays which detect the presence of compounds which bind to CRF₂ receptors, the present invention also provides methods for detecting both CRF₂ receptor agonists and CRF₂ receptor antagonists. Within the context of the present invention, CRF₂ receptor agonists should be understood to refer to molecules that are capable of binding to the cell-surface receptor, thereby stimulating a response pathway within the cell. In contrast, CRF₂ receptor antagonists should be understood to refer to molecules that are capable of binding to a CRF₂ receptor, but which prevent stimulation, or exhibit greatly reduced stimulation of a response pathway within the cell. Various assays may be utilized given the disclosure provided herein in order to screen or select for CRF agonists and antagonists.

For example, within one aspect of the present invention, methods are provided for determining whether a selected compound is a CRF₂ receptor agonist or antagonist, comprising the steps of (a) exposing a selected compound to cells which express CRF₂ receptors under conditions and for a time sufficient to allow binding of the compound and an associated response in intracellular levels of cAMP, and (b) detecting either an increase or decrease in the level of intracellular cAMP, and thereby determining whether the selected compound is a CRF₂ receptor agonist or antagonist.

cAMP may be readily measured using methods which are well known in the art, including, for example, methods described by Salomon et al. (Anal. Biochem. 58:541-548, 1976) or Krishna et al. (J. Pharmacol. Exp. Ther. 163:379, 1968), or, preferably, using commercially available kits such as the Scintillation Proximity Assay Kit from Amersham Corporation, or the ¹²⁵ I RIA cAMP determination kit from Incstar Corporation (Stillwater, Minn.). The Scintillation Proximity Assay Kit measures the production of cAMP by competition of iodinated-cAMP with anti-cAMP antibodies.

Within other aspects, methods are provided for detecting the presence of a CRF₂ receptor agonist or antagonist in a pool of compounds, comprising the steps of (a) exposing a pool of compounds to cells which express CRF₂ receptors under conditions and for a time sufficient to allow binding of the compound and an associated response in intracellular levels of cAMP, and (b) isolating compounds which either increase or decrease the intracellular level of cAMP, such that the presence of a CRF₂ receptor agonist or antagonist may be detected.

Within another aspect, methods for determining whether a selected compound is a CRF₂ receptor antagonist are provided, comprising the steps of (a) exposing a selected compound in the presence of a CRF₂ receptor agonist to a recombinant CRF₂ receptor coupled to a response pathway under conditions and for a time sufficient to allow binding of the compound to the receptor and an associated response through the pathway, and (b) detecting a reduction in the stimulation of the response pathway resulting from the binding of the compound to the CRF₂ receptor, relative to the stimulation of the response pathway by the CRF₂ receptor agonist alone, and therefrom determining the presence of a CRF₂ antagonist. Within other aspects, methods are provided for determining whether a selected compound is a CRF₂ receptor agonist, comprising the steps of (a) exposing a selected compound to a recombinant CRF₂ receptor coupled to a response pathway under conditions and for a time sufficient to allow binding of the compound to the receptor and an associated response through the pathway, and (b) detecting an increase in stimulation of the response pathway resulting from the binding of the compound to the CRF₂ receptor, and therefrom determining the presence of a CRF₂ receptor agonist.

Within particularly preferred embodiments of the invention, the response pathway is a membrane-bound adenylate cyclase response pathway, and the step of detecting comprises measuring the increase or decrease in cAMP production by the membrane-bound adenylate cyclase response pathway. Adenylate cyclase activity assays may be carried out, for example, utilizing method(s) described below in the Examples. Generally, such methods measure the level of stimulation of cAMP relative to known agonists (e.g., sauvagine, urotensin I or CRF), and generally involve exposing a preparation of cells which express a biologically active CRF₂ receptor to the selected compound in the presence of radiolabelled ATP.

Within a further embodiment of the invention, the response pathway includes a reporter system, such as luciferase, or β-galactosidase (see Konig et al., Mol. and Cell. Neuro. 2:331-337, 1991). For examples, within one embodiment of the invention an expression vector is provided comprising a cyclic AMP response element such as a proenkephalin cyclic AMP response element, operably linked to β-galactosidase or luciferase cDNA. The expression vector is then stably transfected into a host cell, and the host cell then transfected with a second expression vector which expresses a CRF₂ receptor. Upon activation of the response pathway, elevated cAMP levels induce the expression of the reporter product, such as the β-galactosidase or luciferase. If the reporter product is luciferase, it is then exposed to luciferin, and photons which are released during the oxidation of luciferin by the luciferase are measured.

A variety of compounds may be screened utilizing such methods. Representative examples include blocking antibodies discussed above, CRF₂ receptor peptides, and CRF analogs (including both peptide and non-peptide ligands). In addition, large numbers of CRF analogs may be generated by saturation mutagenesis of nucleic acid sequences encoding CRF (e.g., Little, Gene 88:113-115, 1990; Hembers et al., Gene 88:143-151, 1989), by segment-directed mutagenesis (e.g., Shortle et al., Proc. Natl Acad. Sci. USA 77:5375-5379, 1980), by forced nucleotide misincorporation (e.g., Liao and Wise, Gene 88:107-111, 1990), or by use of randomly mutagenized oligonucleotides (Hutchison et al., Proc. Natl. Acad. Sci. USA 83:710-714, 1986). Individual transformants expressing a CRF₂ analog may then be cloned as discussed above, or pooled.

Other compounds which may also be screened utilizing methods of the present invention, include chemical compounds which are known and either available commercially (e.g., from Sigma Chemical Co., St. Louis, Mo.) or readily synthesized by one of skill in the art. In addition, numerous novel compositions which are provided in combinatorial libraries may also be readily screened utilizing the methods described herein, including for example organic and protein or peptide libraries (see e.g., Gold and Tuerk, U.S. Pat. No. 5,270,163; and Ladner et al., U.S. Pat. Nos. 5,096,815, 5,198,346, and 5,223,409).

Within a preferred aspect of the invention, the screening of chemical libraries (including peptide, small organic molecule or combinatorial chemistry-derived compound libraries) can be assessed in a high-throughput format using the expressed CRF₂ receptors. More specifically, within one embodiment of the invention, a high throughput receptor binding assay may be performed in a 96-well plate and is automated using the BIOMEK 2000 (Beckman, Fullerton Calif.). Briefly, 1×10⁵ cells transfected with the CRF₂ receptor are grown in each well. To this, 0.05 ml of assay buffer (Dulbecco's Phosphate Buffered Saline, 10 mM MgCl₂, 20 μM Bacitracin) with or without unlabeled r/hCRF (final concentration, 1 μM) is added in duplicate to determine total binding and non-specific binding. In singlets, 0.05 ml of compounds (mixtures from 1 to 30 compounds) are added to each well in addition to 0.05 ml of either ¹²⁵ I!-oCRF or ¹²⁵ I!-r/hCRF (final concentration ˜200 pM). The mixture is incubated for 2 hours at 22° C. Since the transfected cells are adherent, a centrifugation step to separate membrane-bound CRF is not necessary. Next, the fluid is aspirated and the cells are washed three times with approximately 0.9 ml of PBS. After the third wash and aspiration, 0.2 ml of 4M guanidine thiocyanate is added to each well to solubilize the tissue. An aliquot (0.15 ml) of the solubilized sample is monitored in a gamma counter for radioactivity at approximately 80% efficiency. Compounds which demonstrate ≧50% inhibition of ¹²⁵ I!CRF binding are tested in a full dose response competition assay to determine the affinity of these compounds (Ki value) at the CRF₂ receptor.

Once purified partially, or to homogeneity, as desired, both CRF₂ receptor agonists and antagonists may be used therapeutically. Generally substantially pure recombinant CRF antagonists of at least about 50% homogeneity are preferred, at least about 70%-80% homogeneity more preferred, and 95%-99% or more homogeneity most preferred, particularly for pharmaceutical uses. In general, the antagonists may be administered intradermally ("i.d."), intracranially ("i.c."), intraperitoneally ("i.p."), intrathecally ("i.t."), intravenously ("i.v."), subcutaneously ("s.c."), intramuscularly ("i.m.") or directly into a tumor. Typically, the antagonists are present as free bases or as acid salts. Suitable salts should be pharmaceutically acceptable. Representative examples include metal salts, alkali and alkaline earth metal salts such as potassium or sodium salts. Other pharmaceutically acceptable salts include citric, succinic, lactic, hydrochloric and hydrobromic acids. Parenteral compositions may be formulated in aqueous isotonic solutions of between pH 5.6 and 7.4. Suitable isotonic solutions may include sodium chloride, dextrose, boric acid sodium tartrate, and propylene glycol solutions.

LABELS

The nucleic acid molecules, antibodies, CRF₂ receptors, and CRF₂ receptor agonists and antagonists of the present invention may be labeled or conjugated (either through covalent or non-covalent means) to a variety of labels or other molecules, including for example, fluorescent markers, enzyme markers, toxic molecules, molecules which are nontoxic but which become toxic upon exposure to a second compound, and radionuclides.

Representative examples of fluorescent labels suitable for use within the present invention include, for example, Fluorescein Isothiocyanate (FITC), Rodamine, Texas Red, Luciferase and Phycoerythrin (PE). Particularly preferred for use in flow cytometry is FITC which may be conjugated to purified antibody according to the method of Keltkamp in "Conjugation of Fluorescein Isothiocyanate to Antibodies. I. Experiments on the Conditions of Conjugation," Immunology 18:865-873, 1970. (See also Keltkamp, "Conjugation of Fluorescein Isothiocyanate to Antibodies. II. A Reproducible Method," Immunology 18:875-881, 1970; and Goding, "Conjugation of Antibodies with Fluorochromes: Modification to the Standard Methods," J. Immunol. Methods 13:215-226, 1970.) For histochemical staining, HRP, which is preferred, may be conjugated to the purified antibody according to the method of Nakane and Kawaoi ("Peroxidase-Labeled Antibody: A New Method of Conjugation," J. Histochem. Cytochem. 22:1084-1091, 1974; see also, Tijssen and Kurstak, "Highly Efficient and Simple Methods for Preparation of Peroxidase and Active Peroxidase Antibody Conjugates for Enzyme Immunoassays," Anal. Biochem. 136:451-457, 1984).

Representative examples of enzyme markers or labels include alkaline phosphatase, horse radish peroxidase, and β-galactosidase. Representative examples of toxic molecules include ricin, abrin, diphtheria toxin, cholera toxin, gelonin, pokeweed antiviral protein, tritin, Shigella toxin, and Pseudomonas exotoxin A. Representative examples of molecules which are nontoxic, but which become toxic upon exposure to a second compound include thymidine kinases such as HSVTK and VZVTK. Representative examples of radionuclides include Cu-64, Ga-67, Ga-68, Zr-89, Ru-97, Tc-99m, Rh-105, Pd-109, In-111, I-123, I-125, I-131, Re-186, Re-188, Au-198, Au-199, Pb-203, At-211, Pb-212 and Bi-212.

As will be evident to one of skill in the art given the disclosure provided herein, the above described nucleic acid molecules, antibodies, CRF₂ receptors, CRF₂ receptor peptides and CRF₂ receptor agonists and antagonists may also be labeled with other molecules such as colloidal gold, as well either member of a high affinity binding pair (e.g., avidin-biotin).

DIAGNOSTIC USE OF CRF₂ RECEPTOR SEQUENCES

Within another aspect of the present invention, probes and primers are provided for detecting CRF₂ receptors. Within one embodiment of the invention, probes are provided which are capable of hybridizing to CRF₂ receptor nucleic acid or RNA. For purposes of the present invention, probes are "capable of hybridizing" to CRF₂ receptor nucleic acid if they hybridize to Sequence I.D. Nos. 1 or 3 under conditions of moderate or high stringency (see Sambrook et al., supra); but not to CRF receptor nucleic acids. Preferably, the probe may be utilized to hybridize to suitable nucleotide sequences in the presence of 50% formamide, 5×SSPE, 5×Denhardt's, 0.1% SDS and 100 ug/ml Salmon Sperm nucleic acid at 42° C., followed by a first wash with 2×SSC at 42° C., and a second wash with 0.2×SSC at 55° to 60° C.

Probes of the present invention may be composed of either deoxyribonucleic acids (DNA) ribonucleic acids (RNA), nucleic acid analogues, or any combination of these, and may be as few as about 12 nucleotides in length, usually about 14 to 18 nucleotides in length, and possibly as large as the entire sequence of the CRF₂ receptor. Selection of probe size is somewhat dependent upon the use of the probe. For example, in order to determine the presence of various polymorphic forms of the CRF₂ receptor within an individual, a probe comprising virtually the entire length of the CRF₂ receptor coding sequence is preferred. CRF₂ receptor probes may be utilized to identify polymorphisms linked to the CRF₂ receptor gene (see, for example, Weber, Genomics 7:524-530, 1990; and Weber and May, Amer. J. Hum. Gen. 44:388-396, 1989). Such polymorphisms may be associated with inherited diseases such as diabetes.

Probes may be constructed and labeled using techniques which are well known in the art. Shorter probes of, for example, 12 or 14 bases may be generated synthetically. Longer probes of about 75 bases to less than 1.5 kb are preferably generated by, for example, PCR amplification in the presence of labeled precursors such as ³² P-dCTP, digoxigenin-dUTP, or biotin-dATP. Probes of more than 1.5 kb are generally most easily amplified by transfecting a cell with a plasmid containing the relevant probe, growing the transfected cell into large quantities, and purifying the relevant sequence from the transfected cells (see Sambrook et al., supra).

Probes may be labeled by a variety of markers, including, for example, radioactive markers, fluorescent markers, enzymatic markers, and chromogenic markers. The use of ³² P is particularly preferred for marking or labeling a particular probe.

Probes of the present invention may also be utilized to detect the presence of a CRF₂ receptor mRNA or nucleic acid within a sample. However, if CRF₂ receptors are present in only a limited number, or if it is desired to detect a selected mutant sequence which is present in only a limited number, or if it is desired to clone a CRF₂ receptor from a selected warm-blooded animal, then it may be beneficial to amplify the relevant sequence such that it may be more readily detected or obtained.

A variety of methods may be utilized in order to amplify a selected sequence, including, for example, RNA amplification (see Lizardi et al., Bio/Technology 6:1197-1202, 1988; Kramer et al., Nature 339:401-402, 1989; Lomeli et al., Clinical Chem. 35(9):1826-1831, 1989; U.S. Pat. No. 4,786,600), and nucleic acid amplification utilizing Polymerase Chain Reaction ("PCR") (see U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159) (see also, U.S. Pat. Nos. 4,876,187, and 5,011,769, which describe an alternative detection/amplification system comprising the use of scissile linkages).

Within a particularly preferred embodiment, PCR amplification is utilized to detect or obtain a CRF₂ receptor nucleic acid. Briefly, as described in greater detail below, a nucleic acid sample is denatured at 95° C. in order to generate single stranded nucleic acid. Specific primers, as discussed below, are then annealed at 37° C. to 70° C., depending on the proportion of AT/GC in the primers. The primers are extended at 72° C. with Taq polymerase in order to generate the opposite strand to the template. These steps constitute one cycle, which may be repeated in order to amplify the selected sequence.

Primers for the amplification of a selected sequence should be selected from sequences which are highly specific and form stable duplexes with the target sequence. The primers should also be non-complementary, especially at the 3' end, should not form dimers with themselves or other primers, and should not form secondary structures or duplexes with other regions of nucleic acid. In general, primers of about 18 to 20 nucleotides are preferred, and may be easily synthesized using techniques well known in the art.

THERAPEUTIC USES OF CRF₂ RECEPTOR(S)

CRF₂ receptors (or portions thereof), CRF₂ receptor agonists and antagonists, as well as the nucleic acid sequences which encode these molecules may be utilized in a variety of therapeutic applications. For example, within one embodiment of the invention CRF₂ receptors (or portions thereof which bind a substrate such as CRF, sauvagine, or urotensin I) may be utilized in order to reduce vascular levels of the substrate or high ACTH levels due to an excess of the substrate. Thus, CRF₂ receptors may be utilized in treating diseases which are associated with high cortisol levels, including for example Cushing's Disease, alcoholism, anorexia nervosa, and other related disorders.

Similarly, CRF₂ receptors, (or portions thereof which bind a substrate as discussed above) may be utilized in treating tumors which produce high levels of a substrate (e.g., CRF) such as pituitary tumors, as well as for treating abnormalities during pregnancy such as preclampsia which are associated with increased CRF levels. Similarly, they may be utilized to treat hypotension, to modulate the effect of immune system disorders such as arthritis, and to modulate the effect of pituitary disorders. In addition, CRF₂ receptors, (or portions thereof) may be utilized in order to modulate a variety of brain functions, including for example control of satiety, reproduction growth, anxiety, depression, fever, metabolism.

Within yet another aspect of the present invention, viral vectors are provided which may be utilized to treat diseases wherein either the CRF₂ receptor (or a mutant CRF₂ receptor) is over-expressed, or where no CRF₂ receptor is expressed. Briefly, within one embodiment of the invention, viral vectors are provided which direct the production of antisense CRF₂ receptor RNA, in order to prohibit the over-expression of CRF₂ receptors, or the expression of mutant CRF₂ receptors. Within another embodiment, viral vectors are provided which direct the expression of CRF₂ receptor cDNA. Viral vectors suitable for use in the present invention include, among others, recombinant vaccinia vectors (U.S. Pat. Nos. 4,603,112 and 4,769,330), recombinant pox virus vectors (PCT Publication No. WO 89/01973), and preferably, recombinant retroviral vectors ("Recombinant Retroviruses with Amphotropic and Ecoptropic Host Ranges," PCT Publication No. WO 90/02806; "Retroviral Packaging Cell Lines and Processes of Using Same," PCT Publication No. WO 89/07150; and "Antisense RNA for Treatment of Retroviral Disease States," PCT Publication No. WO/03451), and herpesvirus vectors (Kit, Adv. Exp. Med. Biol. 215:219-236, 1989; U.S. Pat. No.5,288,641).

Within various embodiments of the invention, the above-described compositions may be administered in vivo, or ex vivo. Representative routes for in vivo administration include intradermally ("i.d."), intracranially ("i.c."), intraperitoneally ("i.p."), intrathecally ("i.t."), intravenously ("i.v."), subcutaneously ("s.c."), intramuscularly ("i.m.") or even directly into a tumor.

Within other embodiments of the invention, the vectors which contain or express nucleic acid molecules of the present invention, or even the nucleic acid molecules themselves, may be administered by a variety of alternative techniques, including for example direct nucleic acid injection (Acsadi et al., Nature 352:815-818, 1991); microprojectile bombardment (Williams et al., PNAS 88:2726-2730, 1991); liposomes (Pickering et al., Circ. 89(1):13-21, 1994; and Wang et al., PNAS 84:7851-7855, 1987); lipofection (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, 1989); nucleic acid ligand (Wu et al., J. of Biol. Chem. 264:16985-16987, 1989); administration of nucleic acid linked to killed adenovirus (Michael et al., J. Biol. Chem. 268(10):6866-6869, 1993; and Curiel et al., Hum. Gene Ther. 3(2):147-154, 1992), retrotransposons, cytofectin-mediated introduction (DMRIE-DOPE, Vical, Calif.) and transferrin-nucleic acid complexes (Zenke).

The following examples are offered by way of illustration, and not by way of limitation.

EXAMPLES Example 1 ISOLATION OF CRF₂ RECEPTOR cDNA

A. Isolation of CRF₂ receptor cDNA from a rat brain cDNA library

Male Sprague-Dawley rats (Madison, Wis.) weighing between 175-250 gm are decapitated, and the brain excised. Total RNA is then isolated from the brain utilizing a Promega RNAgents Total RNA Kit (catalog #Z5110, Promega, Wisc.) according to the manufacturers instructions, followed by the isolation of poly A+ RNA utilizing a Promega PolyATract kit (catalog #Z5420). A cDNA phage library is then prepared utilizing a Giga-Pack Gold library construction kit according to the manufacturers instructions (catalog #237611, Stratagene, LaJolla, Calif.), which is in turn plated and screened essentially as described by Sambrook et al., (Molecular Cloning) with oligonucleotide 5'-CCCGGATGCC TACAGAGAAT GCCTGGAGGA TGGGACCTGG GCC TCAAGG G-3' (Sequence I.D. No. 5). This oligonucleotide is complementary to nucleotides 440-490 of the rat CRF₂ receptor cDNA sequence shown in FIG. 1.

The phage library is rescreened until a single pure phage isolate is obtained. The phage is then grown on bacterial host XL1-Blue (Stratagene, LaJolla, Calif.), and plasmid nucleic acid is excised with ExAssist helper phage (Stratagene) in SOLR cells. The SOLR cells are then plated, and plasmid nucleic acid is isolated and sequenced utilizing the Sanger dideoxy protocol.

A rat CRF₂ receptor cDNA sequence that may be obtained utilizing this procedure is set forth below in FIG. 2 and in Sequence I.D. No. 1.

B. Isolation of CRF₂ receptor cDNA from a rat hypothalamus cDNA library

CRF2 receptor cDNA can also be isolated from commercially available rat hypothalamus cDNA libraries. Briefly, two million plaques from a rat hypothalamus phage library (Stratagene, catalog #936518) are plated according to the manufacturers instructions, and screened with oligonucleotide Sequence I.D. No. 5 essentially as described above.

A rat CRF₂ receptor cDNA sequence that may be obtained utilizing this procedure is set forth below in FIG. 1 and in Sequence I.D. No. 3.

C. Isolation of CRF₂ receptor cDNA from a human cDNA library

CRF₂ receptor cDNA can also be isolated from commercially available human cDNA libraries. Briefly, approximately two million plaques from a human frontal cortex phage library (Stratagene, catalog #936212) are plated according to the manufacturers instructions, and screened with oligonucleotide 5'-GATCAACTAC TCACAGTGTG AGCCCATTTT GGATGACAAG CAGAGGAAGT A-3' (Sequence I.D. No. 6) essentially as described above.

One human CRF₂ receptor cDNA sequence that may be obtained utilizing this procedure is set forth below in Sequence I.D. No. 7. Briefly, the human sequence is 89.1% identical at the nucleotide level and 93.0% identical at the amino acid level to that of the above-described rat CRF₂α receptor (Sequence I.D. No. 3).

Example 2 EXPRESSION OF CRF₂ RECEPTOR cDNA

pCDM7-Amp is first constructed from pCDM8 (Seed, Nature 329:840-842, 1987; Seed and Aruffo, Proc. Natl. Acad. Sci. 84:3365-3369, 1987; Thomsen et al., Cell 63:485-493, 1990; Bernot and Auffray, Proc. Natl. Acad. Sci 88:2550-2554, 1991; Han et al., Nature 349:697-700, 1991) by deletion of the adeno origin of replication, M13 origin of replication and sup F selection marker. An ampicillin resistance marker is added in order to facilitate selection of the plasmid.

A full-length CRF₂ receptor clone in pBluescriptSK- is isolated from the phage clone described above, and cut with EcoRV and XhoI, releasing the insert. The insert is then isolated and ligated to pCDM7-Amp which had been similarly cut. The resulting product is used to transform E. coli DH5α, from which larger quantities of plasmid nucleic acid may be isolated.

COS-7 (ATCC No. CRL 1651) cells are then transfected with pCDM7-Amp containing CRF₂ receptor cDNA (10 ug nucleic acid/10 cm plate of cells) utilizing 400 μg/ml of DEAE-Dextran and 100 μM chloroquine. The cells are transfected for 4 hours, then shocked with 10% DMSO for 2 minutes. The cells are then washed, and grown in DMEM containing 10% Fetal Bovine Serum for 2 days in a 24-well plate.

Example 3 CRF₂ RECEPTOR BINDING ASSAY

MATERIALS

¹²⁵ I-Tyr⁰ -ovine CRF (¹²⁵ I-oCRF; specific activity, 2200 Ci/mmol) is obtained from Du Pont-New England Nuclear (Boston, Mass.). Unlabeled rat/human CRF is purchased from Peninsula Laboratories (Belmont, Calif.). All other standard reagents were purchased from Sigma Chemical Co. (St. Louis, Mo.).

A. CELL MEMBRANE PREPARATION

Transfect cells which express CRF₂ receptors are harvested, washed once in PBS (pH 7.0 at 22° C.), pelleted by centrifugation and stored at -70° C. until use. On the day of assay, frozen pellets are resuspended in 5 ml of ice cold buffer containing 50 mM Tris, 10 mM MgCl₂, 2 mM EGTA pH 7.0 at 22° C. and homogenized using a Polytron (PT3000, Brinkmann Instruments, Westbury, N.Y.) at 27,000 rpm for 20 s. The homogenate is centrifuged at 48,000×g for 10 min at 4° C., the supernatant discarded, and the pellet was re-homogenized in the same volume of buffer and centrifuged again at 48,000×g for 10 min at 4° C. The resulting pellet containing membranes is resuspended in the above buffer to yield a final protein concentration of 300 mg/ml for use in the assay described below. Protein determinations were performed according to the method of Lowry et al. (J. Biol. Chem. 193:265-275, 1951) using bovine serum albumin (BSA) as a standard.

B. CRF RECEPTOR BINDING ASSAY

One hundred microliters of the membrane suspension is added to 1.5 ml (polypropylene microfuge tubes) containing 100 μl of (100-200 pM) ¹²⁵ I-oCRF in incubation buffer (50 mM Tris, 10 mM MgCl₂, 2 mM EGTA, 1.5% BSA, 0.15 mM bacitracin and 1.5% aprotinin) and 100 μl of the incubation buffer. Competing peptides (e.g., r/hCRF, sauvagine, urotensin I, urotensin II, bovine CRF (b-CRF), and deamidated bovine CRF (b-CRF-OH)) are also added. Incubations are carried out at room temperature (22° C.) for 2 hours, and terminated by centrifugation in a microfuge for 5 min at 12,000×g. The resulting pellet is washed gently with 1.0 ml of ice-cold phosphate-buffered saline, pH 7.2 containing 0.01% Triton X-100 and re-centrifuged for 5 min at 12,000×g. The microfuge tubes are cut just above the pellet, placed into 12×75 mm polystyrene tubes, and monitored for radioactivity in a Packard Cobra II gamma counter at approximately 80% efficiency. The non-specific binding of ¹²⁵ I-oCRF to membrane homogenates, is defined in the presence of 1 mM unlabeled CRF.

C. SATURATION CURVE ANALYSIS

For saturation studies, 100 μl ¹²⁵ I-oCRF (50 pM -10 nM final concentration), 100 μl of assay buffer (with or without 1 μM r/hCRF final concentration, to define the non-specific binding) and 100 μl of membrane suspension (as described above) are added in sequence to 1.5 ml polypropylene microfuge tubes. All reactions are carried out for 2 h at 22° C. and terminated by centrifugation for 5 min at 12,000×g. Aliquots of the supernatant are collected to determine the amount of unbound radioligand. The remaining supernatant is aspirated. Pellets are washed gently with ice-cold PBS plus 0.01% Triton X-100, centrifuged again, and monitored for bound radioactivity. Data from saturation curves are analyzed using the non-linear least-squares curve-fitting program LIGAND (Munson and Rodbard, Anal. Biochem 107:220-229, 1980). Non-specific binding is not defined arbitrarily by the investigator, but rather is estimated as an independent variable from the entire data set.

D. COMPETITION CURVE ANALYSIS

For competition studies, 100 μl ¹²⁵ I-oCRF (final concentration 200-300 pM) is incubated along with 100 μl buffer (in the presence of 1 pM to 10 mM of competing ligands) and 100 μl of membrane suspension as prepared above. The reaction is allowed to proceed for 2 h at 22° C. and was terminated by centrifugation as described above. Data from competition curves were also analyzed by the program LIGAND. For each competition curve, estimates of the affinity of the radiolabeled ligand for the CRF receptor (¹²⁵ I-oCRF or ¹²⁵ I-r/hCRF) are obtained in independent saturation experiments. These estimates are constrained during the analysis of the apparent inhibitory constants (K_(i)) for the various related and unrelated peptides tested. Routinely, the data are analyzed using a one and two-site model comparing the "goodness of fit" between the models in order to accurately determine the K_(i). Statistical analyses provided by LIGAND allowed the determination of whether a single-site or multiple-site model should be used. For CRF-related peptides, all data fit a single site model suggesting that the transfected cells contained a single homogeneous population of binding sites with high affinity and the appropriate pharmacological profile of the CRF receptor.

Example 4 cAMP ASSAY

The effect of CRF and related peptides on adenylate cyclase activity in transfected cells may be determined essentially as follows. For agonist testing, compounds are added to wells with buffer only in order to identify compounds with intrinsic activity. For antagonist testing, compounds are placed in the wells along with 1-100 nM CRF to stimulate the adenylate cyclase system. Compounds are then assessed for their ability to inhibit the CRF-stimulated cAMP production in the transfected cells. Briefly, cells are plated and grown in DMEM containing 10% fetal bovine serum for 2-4 days in 24-well plates at 37° C. On the day of assay, the medium is removed by vacuum aspiration and 100 μl of DMEM, 10 mM MgCl₂, 1 mM isobutylmethylxanthine (a phosphodiesterase inhibitor to inhibit the breakdown of cAMP produced) and 0.1% BSA (pH 7.2) are added to each well. CRF, related peptides and organic test molecules are added to individual wells. The plates are incubated at 37° C. for a period of 1 hour. Following incubation, the wells are aspirated, rinsed once with PBS and aspirated again. 300 μl of 95% Ethanol and 20 mM HCl (EtOH/HCl) are then added to each well and the plates incubated at -20° C. overnight to extract the produced cAMP. The EtOH/HCl is removed, placed in 1.5 ml polypropylene eppendorf tubes and the wells washed with an additional 200 μl of EtOH/HCl and pooled with the initial sample. All samples are dried in a Speed-Vac (Savant Instruments, Farmingdale N.Y.) and either stored at -20° C. until use or reconstituted with 500 μl of sodium acetate buffer pH 7.5 and assayed immediately for cAMP concentration using a radioimmunoassay kit from Biomedical Technologies Inc. (Stoughton Mass.) according to manufacturers instructions.

FIG. 3 represents cAMP accumulation in cells transfected with the CRF1 receptor and stimulated with ovine CRF (oCRF), rat/human CRF (r/hCRF), sauvagine or urotensin I. Briefly, this figure shows a dose-dependent increase in cAMP in response to these compounds. All of these compounds showed similar potencies. In FIG. 4, the rat CRF₂α receptor has been transfected into cells and cAMP accumulation is measured in response to the same drugs as shown in FIG. 3. As shown in FIG. 4, sauvagine and urotensin I are more potent than either oCRF or r/hCRF at stimulating cAMP. Together, these findings show a clearly distinct pharmacological profile between the CRF₁ and CRF₂ receptors.

Example 5 TISSUE DISTRIBUTION OF CRF₂ RECEPTORS

In order to determine anatomical distribution of the two CRF₂ receptor forms, mRNA expression patterns were analyzed in isolated RNA (RNase protection assays) and whole tissue sections (in situ hybridization) from rat brain and peripheral tissues essentially as described below.

A. RNase Protection Assay

A 278-base riboprobe, containing 181-base antisense sequence specific to rat the CRF₂α receptor, and a 366-base riboprobe containing 258-base antisense sequence specific to the CRF₂β receptor mRNA were generated by T3 RNA polymerase in 40 mM Tris-HCl (pH 7.5), 6 mM MgCl, 2 mM spermidine, 10 mM NaCl, 10 mM DTT, 1 U/μl of RNasin (Promega), 0.5 mM each of ATP, CTP, TTP, and 3 μM of ³² P-UTP (DuPont, 800 Ci/mmol), 0.1 μg/μl of template DNA, 1 U/μl of T3 RNA polymerase in total volume of 10 μl at 37° C. for 30 minutes. A 712-base riboprobe containing 632-base of antisense sequence specific to rat β-actin mRNA was generated in the same conditions, except using T7 RNA polymerase. The probe synthesis mixtures were then treated with DNase I at concentration of 1 U/μl at 37° C. for 10 minutes and then electrophoresed on a 5% denaturing acrylamide gel. The ³² P-labeled RNA probes with expected sizes were recovered from the gel with an RNA purification kit, RNaid kit (Bio-101, La Jolla, Calif.). RNA hybridization, RNase digestion and separation of protected RNAs were performed as described by Sawbrook et al., supra. In assays for CRF₂ receptor mRNAs, 40 μg of total RNA was used in each sample. In assays for β-actin mRNA, 1 μg of total RNA was used for each sample.

B. In Situ Hybridization

Male Sprague-Dawley rats were sacrificed by decapitation, and the brains removed and frozen in liquid isopentane (-42° C.). Subsequently, tissues were sectioned (15 μm) on a cryostat maintained at -20° C. and thaw mounted onto polylysine-coated microscope slides. Sections were stored at -80° C. prior to tissue fixation.

Sections were removed from storage at -80° C. and placed directly into 4% buffered paraformaldehyde at room temperature. After 60 minutes, slides were rinsed in isotonic phosphate buffered saline (10 minutes) and treated with proteinase K (1 μg/ml) in 100 mM Tris/HCl, pH 8.0) for 10 minutes at 37° C. Subsequently, sections underwent successive washes in water (1 minute), 0.1M triethanolamine (pH 8.0, plus 0.25% acetic anhydride) for 10 minutes and 2×SSC (0.3 mM NaCl, 0.03 mM sodium citrate, pH 7.2) for 5 minutes. Sections were then dehydrated through graded alcohols and air dried.

RNA probes were synthesized using Maxiscript RNA transcription kits (Ambion, Austin, Tex.). Post-fixed sections were hybridized with 1.0×10⁶ dpm of ³⁵ S!UTP-labeled riboprobes in hybridization buffer containing 75% formamide, 10% dextran sulphate, 3×SSC, 50 mM sodium phosphate buffer (pH 7.4), I×Denhardt's solution, 0.1 mg/ml yeast tRNA and 10 mM dithiothreitol in a total volume of 25 μl. The diluted probe was applied to sections on a glass coverslip which was sealed into place with rubber cement. Sections were hybridized overnight at 55° C. in a humid environment.

Post-hybridization the rubber cement was removed and sections were washed in 2×SSC for 5 minutes and then treated with RNase A (200 μg/ml in 10 mM Tris/HCL, pH 8.0, containing 0.5M NaCl) for 60 minutes at 37° C. Subsequently, sections were washed in 2×SSC for 5 minutes, 1×SSC for 5 minutes, 0.5×SSC for 60 minutes at hybridization temperature, 0.5×SSC for 60 minutes at room temperature for 5 minutes and then dehydrated in graded alcohols and air dried. For signal detection, sections were placed on Kodak XAR-5 X-ray film and exposed for 7 days at room temperature.

C. Results

The CRF₂α and CRF₂β receptor mRNAs have clearly distinct tissue distributions. In particular, CRF₂α is found primarily in the brain, particularly in the hypothalamus, lateral septum and olfactory bulb, whereas the CRF₂β mRNA is found primarily, but not restricted to, the heart and skeletal muscle. This is in stark contrast to the previously disclosed distribution of the CRF₁ receptor. (Potter et al., PNAS 91:8777-8781, 1994).

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 8                                                   (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1514 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 44..1336                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        GATCCCTATCCCTGAGCAAGCGAGTGGCAGGATCTGGTGTCCCATGGGGCACCCA55                      MetGlyHisPro                                                                   GGCTCTCTTCCCAGTGCACAACTCCTCCTCTGCCTATACTCTCTGCTC103                            GlySerLeuProSerAlaGlnLeuLeuLeuCysLeuTyrSerLeuLeu                               5101520                                                                        CCACTGCTCCAGGTGGCCCAACCAGGCAGGCCACTCCAGGACCAGCCC151                            ProLeuLeuGlnValAlaGlnProGlyArgProLeuGlnAspGlnPro                               253035                                                                         CTGTGGACACTTTTGGAGCAGTACTGCCATAGGACCACAACTCGGAAT199                            LeuTrpThrLeuLeuGluGlnTyrCysHisArgThrThrThrArgAsn                               404550                                                                         TTTTCAGGTCCCTACTCCTACTGCAACACGACCTTGGACCAGATCGGG247                            PheSerGlyProTyrSerTyrCysAsnThrThrLeuAspGlnIleGly                               556065                                                                         ACCTGCTGGCCCCAGAGCGCGCCTGGAGCCCTAGTGGAGAGACCATGC295                            ThrCysTrpProGlnSerAlaProGlyAlaLeuValGluArgProCys                               707580                                                                         CCCGAATACTTCAACGGCATCAAGTACAACACGACCCGGAATGCCTAC343                            ProGluTyrPheAsnGlyIleLysTyrAsnThrThrArgAsnAlaTyr                               859095100                                                                      AGAGAATGCCTGGAGAATGGGACCTGGGCCTCAAGGATCAACTACTCA391                            ArgGluCysLeuGluAsnGlyThrTrpAlaSerArgIleAsnTyrSer                               105110115                                                                      CACTGTGAACCCATTTTGGATGACAAGCAGAGGAAGTATGACCTGCAT439                            HisCysGluProIleLeuAspAspLysGlnArgLysTyrAspLeuHis                               120125130                                                                      TACCGAATCGCCCTCATCATCAACTACCTGGGCCACTGTGTTTCCGTG487                            TyrArgIleAlaLeuIleIleAsnTyrLeuGlyHisCysValSerVal                               135140145                                                                      GTGGCCCTGGTGGCTGCTTTCCTGCTTTTCCTAGTGCTGCGGAGTATC535                            ValAlaLeuValAlaAlaPheLeuLeuPheLeuValLeuArgSerIle                               150155160                                                                      CGCTGCCTGCGGAATGTGATCCACTGGAACCTCATCACCACCTTCATC583                            ArgCysLeuArgAsnValIleHisTrpAsnLeuIleThrThrPheIle                               165170175180                                                                   CTGAGAAACATCACGTGGTTCCTGCTGCAACTCATCGACCACGAAGTG631                            LeuArgAsnIleThrTrpPheLeuLeuGlnLeuIleAspHisGluVal                               185190195                                                                      CATGAGGGCAATGAGGTCTGGTGCCGCTGCGTCACCACCATATTCAAC679                            HisGluGlyAsnGluValTrpCysArgCysValThrThrIlePheAsn                               200205210                                                                      TACTTTGTGGTCACCAACTTCTTCTGGATGTTTGTGGAAGGCTGCTAC727                            TyrPheValValThrLysPhePheTrpMetPheValGluGlyCysTyr                               215220225                                                                      CTGCACACGGCCATCGTCATGACGTACTCCACGGAGCATCTGCGCAAG775                            LeuHisThrAlaIleValMetThrTyrSerThrGluHisLeuArgLys                               230235240                                                                      TGGCTCTTCCTCTTCATTGGATGGTGCATACCCTGCCCTATCATTGTC823                            TrpLeuPheLeuPheIleGlyTrpCysIleProCysProIleIleVal                               245250255260                                                                   GCCTGGGCAGTTGGCAAACTCTACTATGAGAATGAGCAGTGCTGGTTT871                            AlaTrpAlaValGlyLysLeuTyrTyrGluAsnGluGlnCysTrpPhe                               265270275                                                                      GGCAAGGAACCTGGTGACTTAGTGGACTACATCTACCAGGGCCCCATC919                            GlyLysGluProGlyAspLeuValAspTyrIleTyrGlnGlyProIle                               280285290                                                                      ATCCTCGTGCTCCTCATCAATTTTGTGTTTCTGTTCAACATCGTCAGG967                            IleLeuValLeuLeuIleAsnPheValPheLeuPheAsnIleValArg                               295300305                                                                      ATCCTGATGACAAAACTGCGAGCCTCCACCACATCCGAGACCATCCAG1015                           IleLeuMetThrLysLeuArgAlaSerThrThrSerGluThrIleGln                               310315320                                                                      TACAGGAAGGCAGTGAAGGCCACCCTGGTCCTCCTCCCCCTGTTGGGC1063                           TyrArgLysAlaValLysAlaThrLeuValLeuLeuProLeuLeuGly                               325330335340                                                                   ATCACCTACATGCTCTTCTTTGTCAATCCTGGAGAGGACGACCTGTCA1111                           IleThrTyrMetLeuPhePheValAsnProGlyGluAspAspLeuSer                               345350355                                                                      CAGATTGTGTTCATCTACTTCAACTCTTTCCTGCAGTCCTTTCAGGGT1159                           GlnIleValPheIleTyrPheAsnSerPheLeuGlnSerPheGlnGly                               360365370                                                                      TTCTTTGTGTCCGTTTTCTACTGCTTCTTCAATGGAGAGGTGCGCTCC1207                           PhePheValSerValPheTyrCysPhePheAsnGlyGluValArgSer                               375380385                                                                      GCCCTGAGAAAGCGGTGGCACCGTTGGCAGGACCACCACGCCCTCCGA1255                           AlaLeuArgLysArgTrpHisArgTrpGlnAspHisHisAlaLeuArg                               390395400                                                                      GTGCCTGTGGCCCGGGCCATGTCCATTCCCACATCGCCCACCAGGATC1303                           ValProValAlaArgAlaMetSerIleProThrSerProThrArgIle                               405410415420                                                                   AGCTTCCACAGCATCAAGCAGACAGCTGCCGTGTGATCCCCTGTCACCCATCT1356                      SerPheHisSerIleLysGlnThrAlaAlaVal                                              425430                                                                         GCCCAGCACTCCACCACCGAGGCGGCTTCCTCATTCTTCACAGCCTTCCCTGGGTCCTCC1416               TTGCTACACTGACCCTTGGGTGCAGGAGAAGGGGGGGTGGATGAACTCTCCTGCCGGAAG1476               AAAGGAAAACTATGAAATGGAGGCTCTGAAAGACCAGG1514                                     (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 431 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                        MetGlyHisProGlySerLeuProSerAlaGlnLeuLeuLeuCysLeu                               151015                                                                         TyrSerLeuLeuProLeuLeuGlnValAlaGlnProGlyArgProLeu                               202530                                                                         GlnAspGlnProLeuTrpThrLeuLeuGluGlnTyrCysHisArgThr                               354045                                                                         ThrThrArgAsnPheSerGlyProTyrSerTyrCysAsnThrThrLeu                               505560                                                                         AspGlnIleGlyThrCysTrpProGlnSerAlaProGlyAlaLeuVal                               65707580                                                                       GluArgProCysProGluTyrPheAsnGlyIleLysTyrAsnThrThr                               859095                                                                         ArgAsnAlaTyrArgGluCysLeuGluAsnGlyThrTrpAlaSerArg                               100105110                                                                      IleAsnTyrSerHisCysGluProIleLeuAspAspLysGlnArgLys                               115120125                                                                      TyrAspLeuHisTyrArgIleAlaLeuIleIleAsnTyrLeuGlyHis                               130135140                                                                      CysValSerValValAlaLeuValAlaAlaPheLeuLeuPheLeuVal                               145150155160                                                                   LeuArgSerIleArgCysLeuArgAsnValIleHisTrpAsnLeuIle                               165170175                                                                      ThrThrPheIleLeuArgAsnIleThrTrpPheLeuLeuGlnLeuIle                               180185190                                                                      AspHisGluValHisGluGlyAsnGluValTrpCysArgCysValThr                               195200205                                                                      ThrIlePheAsnTyrPheValValThrLysPhePheTrpMetPheVal                               210215220                                                                      GluGlyCysTyrLeuHisThrAlaIleValMetThrTyrSerThrGlu                               225230235240                                                                   HisLeuArgLysTrpLeuPheLeuPheIleGlyTrpCysIleProCys                               245250255                                                                      ProIleIleValAlaTrpAlaValGlyLysLeuTyrTyrGluAsnGlu                               260265270                                                                      GlnCysTrpPheGlyLysGluProGlyAspLeuValAspTyrIleTyr                               275280285                                                                      GlnGlyProIleIleLeuValLeuLeuIleAsnPheValPheLeuPhe                               290295300                                                                      AsnIleValArgIleLeuMetThrLysLeuArgAlaSerThrThrSer                               305310315320                                                                   GluThrIleGlnTyrArgLysAlaValLysAlaThrLeuValLeuLeu                               325330335                                                                      ProLeuLeuGlyIleThrTyrMetLeuPhePheValAsnProGlyGlu                               340345350                                                                      AspAspLeuSerGlnIleValPheIleTyrPheAsnSerPheLeuGln                               355360365                                                                      SerPheGlnGlyPhePheValSerValPheTyrCysPhePheAsnGly                               370375380                                                                      GluValArgSerAlaLeuArgLysArgTrpHisArgTrpGlnAspHis                               385390395400                                                                   HisAlaLeuArgValProValAlaArgAlaMetSerIleProThrSer                               405410415                                                                      ProThrArgIleSerPheHisSerIleLysGlnThrAlaAlaVal                                  420425430                                                                      (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1626 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 216..1449                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                        GCGGCCCCTCATCTCCGTGAGCCCCGAGGCTTCTCTTGGCCAAGGTCCTAGGAGTGATCC60                 GATTGAGAGCGGCGCCCCAAAGCTGCCGGGCTGGCCGGGGTGGGCGGGGAGGCACCTGGA120                CGCTGCACTCTCTGGTGGCTCCGCGTCGCGCCAGGTCCCTCGCAGCCACGCGGGGCGCGC180                ACTCCCACTCCCAACGCGCGCGGCTCCGGAGCGCAATGGACGCGGCGCTGCTC233                       MetAspAlaAlaLeuLeu                                                             15                                                                             CTCAGCCTGCTGGAGGCCAACTGCAGCCTGGCACTGGCCGAAGAGCTG281                            LeuSerLeuLeuGluAlaAsnCysSerLeuAlaLeuAlaGluGluLeu                               101520                                                                         CTTTTGGACGGCTGGGGAGAGCCCCCGGACCCCGAAGGTCCCTACTCC329                            LeuLeuAspGlyTrpGlyGluProProAspProGluGlyProTyrSer                               253035                                                                         TACTGCAACACGACCTTGGACCAGATCGGGACCTGCTGGCCCCAGAGC377                            TyrCysAsnThrThrLeuAspGlnIleGlyThrCysTrpProGlnSer                               404550                                                                         GCGCCTGGAGCCCTAGTGGAGAGACCATGCCCCGAATACTTCAACGGC425                            AlaProGlyAlaLeuValGluArgProCysProGluTyrPheAsnGly                               55606570                                                                       ATCAAGTACAACACGACCCGGAATGCCTACAGAGAATGCCTGGAGAAT473                            IleLysTyrAsnThrThrArgAsnAlaTyrArgGluCysLeuGluAsn                               758085                                                                         GGGACCTGGGCCTCAAGGATCAACTACTCACACTGTGAACCCATTTTG521                            GlyThrTrpAlaSerArgIleAsnTyrSerHisCysGluProIleLeu                               9095100                                                                        GATGACAAGCAGAGGAAGTATGACCTGCATTACCGAATCGCCCTCATC569                            AspAspLysGlnArgLysTyrAspLeuHisTyrArgIleAlaLeuIle                               105110115                                                                      ATCAACTACCTGGGCCACTGTGTTTCCGTGGTGGCCCTGGTGGCTGCT617                            IleAsnTyrLeuGlyHisCysValSerValValAlaLeuValAlaAla                               120125130                                                                      TTCCTGCTTTTCCTAGTGCTGCGGAGTATCCGCTGCCTGCGGAATGTG665                            PheLeuLeuPheLeuValLeuArgSerIleArgCysLeuArgAsnVal                               135140145150                                                                   ATCCACTGGAACCTCATCACCACCTTCATCCTGAGAAACATCACGTGG713                            IleHisTrpAsnLeuIleThrThrPheIleLeuArgAsnIleThrTrp                               155160165                                                                      TTCCTGCTGCAACTCATCGACCACGAAGTGCATGAGGGCAATGAGGTC761                            PheLeuLeuGlnLeuIleAspHisGluValHisGluGlyAsnGluVal                               170175180                                                                      TGGTGCCGCTGCGTCACCACCATATTCAACTACTTTGTGGTCACCAAC809                            TrpCysArgCysValThrThrIlePheAsnTyrPheValValThrAsn                               185190195                                                                      TTCTTCTGGATGTTTGTGGAAGGCTGCTACCTGCACACGGCCATCGTC857                            PhePheTrpMetPheValGluGlyCysTyrLeuHisThrAlaIleVal                               200205210                                                                      ATGACGTACTCCACGGAGCATCTGCGCAAGTGGCTCTTCCTCTTCATT905                            MetThrTyrSerThrGluHisLeuArgLysTrpLeuPheLeuPheIle                               215220225230                                                                   GGATGGTGCATACCCTGCCCTATCATTGTCGCCTGGGCAGTTGGCAAA953                            GlyTrpCysIleProCysProIleIleValAlaTrpAlaValGlyLys                               235240245                                                                      CTCTACTATGAGAATGAGCAGTGCTGGTTTGGCAAGGAACCTGGTGAC1001                           LeuTyrTyrGluAsnGluGlnCysTrpPheGlyLysGluProGlyAsp                               250255260                                                                      TTAGTGGACTACATCTACCAGGGCCCCATCATCCTCGTGCTCCTCATC1049                           LeuValAspTyrIleTyrGlnGlyProIleIleLeuValLeuLeuIle                               265270275                                                                      AATTTTGTGTTTCTGTTCAACATCGTCAGGATCCTGATGACAAAACTG1097                           AsnPheValPheLeuPheAsnIleValArgIleLeuMetThrLysLeu                               280285290                                                                      CGAGCCTCCACCACATCCGAGACCATCCAGTACAGGAAGGCAGTGAAG1145                           ArgAlaSerThrThrSerGluThrIleGlnTyrArgLysAlaValLys                               295300305310                                                                   GCCACCCTGGTCCTCCTCCCCCTGTTGGGCATCACCTACATGCTCTTC1193                           AlaThrLeuValLeuLeuProLeuLeuGlyIleThrTyrMetLeuPhe                               315320325                                                                      TTTGTCAATCCTGGAGAGGACGACCTGTCACAGATTGTGTTCATCTAC1241                           PheValAsnProGlyGluAspAspLeuSerGlnIleValPheIleTyr                               330335340                                                                      TTCAACTCTTTCCTGCAGTCCTTTCAGGGTTTCTTTGTGTCCGTTTTC1289                           PheAsnSerPheLeuGlnSerPheGlnGlyPhePheValSerValPhe                               345350355                                                                      TACTGCTTCTTCAATGGAGAGGTGCGCTCCGCCCTGAGAAAGCGGTGG1337                           TyrCysPhePheAsnGlyGluValArgSerAlaLeuArgLysArgTrp                               360365370                                                                      CACCGTTGGCAGGACCACCACGCCCTCCGAGTGCCTGTGGCCCGGGCC1385                           HisArgTrpGlnAspHisHisAlaLeuArgValProValAlaArgAla                               375380385390                                                                   ATGTCCATTCCCACATCGCCCACCAGGATCAGCTTCCACAGCATCAAG1433                           MetSerIleProThrSerProThrArgIleSerPheHisSerIleLys                               395400405                                                                      CAGACAGCTGCCGTGTGATCCCCTGTCACCCATCTGCCCAGCACTC1479                             GlnThrAlaAlaVal                                                                410                                                                            CACCACCGAGGCGGCTTCCTCATTCTTCACAGCCTTCCCTGGGTCCTCCTTGCTACACTG1539               ACCCTTGGGTGCAGGAGAAGGGGGGGTGGATGAACTCTCCTGCCGGAAGAAAGGAAAACT1599               ATGAAATGGAGGCTCTGAAAGACCAGG1626                                                (2) INFORMATION FOR SEQ ID NO:4:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 411 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                        MetAspAlaAlaLeuLeuLeuSerLeuLeuGluAlaAsnCysSerLeu                               151015                                                                         AlaLeuAlaGluGluLeuLeuLeuAspGlyTrpGlyGluProProAsp                               202530                                                                         ProGluGlyProTyrSerTyrCysAsnThrThrLeuAspGlnIleGly                               354045                                                                         ThrCysTrpProGlnSerAlaProGlyAlaLeuValGluArgProCys                               505560                                                                         ProGluTyrPheAsnGlyIleLysTyrAsnThrThrArgAsnAlaTyr                               65707580                                                                       ArgGluCysLeuGluAsnGlyThrTrpAlaSerArgIleAsnTyrSer                               859095                                                                         HisCysGluProIleLeuAspAspLysGlnArgLysTyrAspLeuHis                               100105110                                                                      TyrArgIleAlaLeuIleIleAsnTyrLeuGlyHisCysValSerVal                               115120125                                                                      ValAlaLeuValAlaAlaPheLeuLeuPheLeuValLeuArgSerIle                               130135140                                                                      ArgCysLeuArgAsnValIleHisTrpAsnLeuIleThrThrPheIle                               145150155160                                                                   LeuArgAsnIleThrTrpPheLeuLeuGlnLeuIleAspHisGluVal                               165170175                                                                      HisGluGlyAsnGluValTrpCysArgCysValThrThrIlePheAsn                               180185190                                                                      TyrPheValValThrAsnPhePheTrpMetPheValGluGlyCysTyr                               195200205                                                                      LeuHisThrAlaIleValMetThrTyrSerThrGluHisLeuArgLys                               210215220                                                                      TrpLeuPheLeuPheIleGlyTrpCysIleProCysProIleIleVal                               225230235240                                                                   AlaTrpAlaValGlyLysLeuTyrTyrGluAsnGluGlnCysTrpPhe                               245250255                                                                      GlyLysGluProGlyAspLeuValAspTyrIleTyrGlnGlyProIle                               260265270                                                                      IleLeuValLeuLeuIleAsnPheValPheLeuPheAsnIleValArg                               275280285                                                                      IleLeuMetThrLysLeuArgAlaSerThrThrSerGluThrIleGln                               290295300                                                                      TyrArgLysAlaValLysAlaThrLeuValLeuLeuProLeuLeuGly                               305310315320                                                                   IleThrTyrMetLeuPhePheValAsnProGlyGluAspAspLeuSer                               325330335                                                                      GlnIleValPheIleTyrPheAsnSerPheLeuGlnSerPheGlnGly                               340345350                                                                      PhePheValSerValPheTyrCysPhePheAsnGlyGluValArgSer                               355360365                                                                      AlaLeuArgLysArgTrpHisArgTrpGlnAspHisHisAlaLeuArg                               370375380                                                                      ValProValAlaArgAlaMetSerIleProThrSerProThrArgIle                               385390395400                                                                   SerPheHisSerIleLysGlnThrAlaAlaVal                                              405410                                                                         (2) INFORMATION FOR SEQ ID NO:5:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 50 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                        CCCGGATGCCTACAGAGAATGCCTGGAGGATGGGACCTGGGCCTCAAGGG50                           (2) INFORMATION FOR SEQ ID NO:6:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 51 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                        GATCAACTACTCACAGTGTGAGCCCATTTTGGATGACAAGCAGAGGAAGTA51                          (2) INFORMATION FOR SEQ ID NO:7:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1468 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..1233                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                        ATGGACGCGGCACTGCTCCACAGCCTGCTGGAGGCCAACTGCAGCCTG48                             MetAspAlaAlaLeuLeuHisSerLeuLeuGluAlaAsnCysSerLeu                               151015                                                                         GCGCTGGCTGAAGAGCTGCTCTTGGACGGCTGGGGGCCACCCCTGGAC96                             AlaLeuAlaGluGluLeuLeuLeuAspGlyTrpGlyProProLeuAsp                               202530                                                                         CCCGAGGGTCCCTACTCCTACTGCAACACGACCTTGGACCAGATCGGA144                            ProGluGlyProTyrSerTyrCysAsnThrThrLeuAspGlnIleGly                               354045                                                                         ACGTGCTGGCCCCGCAGCGCTGCCGGAGCCCTCGTGGAGAGGCCGTGC192                            ThrCysTrpProArgSerAlaAlaGlyAlaLeuValGluArgProCys                               505560                                                                         CCCGAGTACTTCAACGGCGTCAAGTACAACACGACCCGGAATGCCTAT240                            ProGluTyrPheAsnGlyValLysTyrAsnThrThrArgAsnAlaTyr                               65707580                                                                       CGAGAATGCTTGGAGAATGGGACGTGGGCCTCAAAGATCAACTACTCA288                            ArgGluCysLeuGluAsnGlyThrTrpAlaSerLysIleAsnTyrSer                               859095                                                                         CAGTGTGAGCCCATTTTGGATGACAAGCAGAGGAAGTATGACCTGCAC336                            GlnCysGluProIleLeuAspAspLysGlnArgLysTyrAspLeuHis                               100105110                                                                      TACCGCATCGCCCTTGTCGTCAACTACCTGGGCCACTGCGTATCTGTG384                            TyrArgIleAlaLeuValValAsnTyrLeuGlyHisCysValSerVal                               115120125                                                                      GCAGCCCTGGTGGCCGCCTTCCTGCTTTTCCTGGCCCTGCGGAGCATT432                            AlaAlaLeuValAlaAlaPheLeuLeuPheLeuAlaLeuArgSerIle                               130135140                                                                      CGCTGTCTGCGGAATGTGATTCACTGGAACCTCATCACCACCTTTATC480                            ArgCysLeuArgAsnValIleHisTrpAsnLeuIleThrThrPheIle                               145150155160                                                                   CTGCGAAATGTCATGTGGTTCCTGCTGCAGCTCGTTGACCATGAAGTG528                            LeuArgAsnValMetTrpPheLeuLeuGlnLeuValAspHisGluVal                               165170175                                                                      CACGAGAGCAATGAGGTCTGGTGCCACTGCATCACCACCATCTTCAAC576                            HisGluSerAsnGluValTrpCysHisCysIleThrThrIlePheAsn                               180185190                                                                      TACTTCGTGGTGACCAACTTCTTCTGGATGTTTGTGGAAGGCTGCTAC624                            TyrPheValValThrAsnPhePheTrpMetPheValGluGlyCysTyr                               195200205                                                                      CTGCACACGGCCATTGTCATGACCTACTCCACTGAGCGCCTGCGCAAG672                            LeuHisThrAlaIleValMetThrTyrSerThrGluArgLeuArgLys                               210215220                                                                      TGCCTCTTCCTCTTCATCGGATGGTGCATCCCCTTCCCCATCATCGTC720                            CysLeuPheLeuPheIleGlyTrpCysIleProPheProIleIleVal                               225230235240                                                                   GCCTGGGCCATCGGCAAGCTCTACTATGAGAATGAACAGTGCTGGTTT768                            AlaTrpAlaIleGlyLysLeuTyrTyrGluAsnGluGlnCysTrpPhe                               245250255                                                                      GGCAAGGAGCCTGGCGACCTGGTGGACTACATCTACCAAGGCCCCATC816                            GlyLysGluProGlyAspLeuValAspTyrIleTyrGlnGlyProIle                               260265270                                                                      ATTCTCGTGCTCCTGATCAATTTCGTATTTCTGTTCAACATCGTCAGG864                            IleLeuValLeuLeuIleAsnPheValPheLeuPheAsnIleValArg                               275280285                                                                      ATCCTAATGACAAAGTTACGCGCGTCCACCACATCCGAGACAATCCAG912                            IleLeuMetThrLysLeuArgAlaSerThrThrSerGluThrIleGln                               290295300                                                                      TACAGGAAGGCAGTGAAGGCCACCCTGGTGCTCCTGCCCCTCCTGGGC960                            TyrArgLysAlaValLysAlaThrLeuValLeuLeuProLeuLeuGly                               305310315320                                                                   ATCACCTACATGCTCTTCTTCGTCAATCCCGGGGAGGACGACCTGTCA1008                           IleThrTyrMetLeuPhePheValAsnProGlyGluAspAspLeuSer                               325330335                                                                      CAGATCATGTTCATCTATTTCAACTCCTTCCTGCAGTCGTTCCAGGGT1056                           GlnIleMetPheIleTyrPheAsnSerPheLeuGlnSerPheGlnGly                               340345350                                                                      TTCTTCGTGTCTGTCTTCTACTGCTTCTTCAATGGAGAGGTGCGCTCA1104                           PhePheValSerValPheTyrCysPhePheAsnGlyGluValArgSer                               355360365                                                                      GCCGTGAGGAAGAGGTGGCACCGCTGGCAGGACCATCACTCCCTTCGA1152                           AlaValArgLysArgTrpHisArgTrpGlnAspHisHisSerLeuArg                               370375380                                                                      GTCCCCATGGCCCGGGCCATGTCCATCCCTACATCACCCACACGGATC1200                           ValProMetAlaArgAlaMetSerIleProThrSerProThrArgIle                               385390395400                                                                   AGCTTCCACAGCATCAAGCAGACGGCCGCTGTGTGACCCCTCGGTCGCCCACC1253                      SerPheHisSerIleLysGlnThrAlaAlaVal                                              405410                                                                         TGCACAGCTCCCCTGTCCTCCTCCACCTTCTTCCTCTGGGTTCTCTGTGCTGGGCAGGCT1313               CTCGTGGGGCAGGAGATGGGAGGGGAGAGACCAGCTCTCCAGCCTGGCAGGAAAGAGGGG1373               GTGCGGCAGCCAAGGGGGACTGCAAGGGACAGGGATGAGTGGGGGCCACCAGGCTCAGCG1433               CAAGAGGAAGCAGAGGGAATTCGATGGTGGAGCTC1468                                        (2) INFORMATION FOR SEQ ID NO:8:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 411 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                        MetAspAlaAlaLeuLeuHisSerLeuLeuGluAlaAsnCysSerLeu                               151015                                                                         AlaLeuAlaGluGluLeuLeuLeuAspGlyTrpGlyProProLeuAsp                               202530                                                                         ProGluGlyProTyrSerTyrCysAsnThrThrLeuAspGlnIleGly                               354045                                                                         ThrCysTrpProArgSerAlaAlaGlyAlaLeuValGluArgProCys                               505560                                                                         ProGluTyrPheAsnGlyValLysTyrAsnThrThrArgAsnAlaTyr                               65707580                                                                       ArgGluCysLeuGluAsnGlyThrTrpAlaSerLysIleAsnTyrSer                               859095                                                                         GlnCysGluProIleLeuAspAspLysGlnArgLysTyrAspLeuHis                               100105110                                                                      TyrArgIleAlaLeuValValAsnTyrLeuGlyHisCysValSerVal                               115120125                                                                      AlaAlaLeuValAlaAlaPheLeuLeuPheLeuAlaLeuArgSerIle                               130135140                                                                      ArgCysLeuArgAsnValIleHisTrpAsnLeuIleThrThrPheIle                               145150155160                                                                   LeuArgAsnValMetTrpPheLeuLeuGlnLeuValAspHisGluVal                               165170175                                                                      HisGluSerAsnGluValTrpCysHisCysIleThrThrIlePheAsn                               180185190                                                                      TyrPheValValThrAsnPhePheTrpMetPheValGluGlyCysTyr                               195200205                                                                      LeuHisThrAlaIleValMetThrTyrSerThrGluArgLeuArgLys                               210215220                                                                      CysLeuPheLeuPheIleGlyTrpCysIleProPheProIleIleVal                               225230235240                                                                   AlaTrpAlaIleGlyLysLeuTyrTyrGluAsnGluGlnCysTrpPhe                               245250255                                                                      GlyLysGluProGlyAspLeuValAspTyrIleTyrGlnGlyProIle                               260265270                                                                      IleLeuValLeuLeuIleAsnPheValPheLeuPheAsnIleValArg                               275280285                                                                      IleLeuMetThrLysLeuArgAlaSerThrThrSerGluThrIleGln                               290295300                                                                      TyrArgLysAlaValLysAlaThrLeuValLeuLeuProLeuLeuGly                               305310315320                                                                   IleThrTyrMetLeuPhePheValAsnProGlyGluAspAspLeuSer                               325330335                                                                      GlnIleMetPheIleTyrPheAsnSerPheLeuGlnSerPheGlnGly                               340345350                                                                      PhePheValSerValPheTyrCysPhePheAsnGlyGluValArgSer                               355360365                                                                      AlaValArgLysArgTrpHisArgTrpGlnAspHisHisSerLeuArg                               370375380                                                                      ValProMetAlaArgAlaMetSerIleProThrSerProThrArgIle                               385390395400                                                                   SerPheHisSerIleLysGlnThrAlaAlaVal                                              405410                                                                         __________________________________________________________________________ 

We claim:
 1. An isolated nucleic acid molecule encoding a mammalian CRF₂ receptor, wherein said nucleic acid molecule comprises:(a) a nucleic acid molecule which encodes an amino acid sequence selected from the group consisting of Sequence I.D. Nos. 2, 4, and 8; or (b) a nucleic acid molecule which hybridizes under conditions of moderate stringency to the complement of a nucleic acid molecule which encodes Sequence I.D. Nos. 2, 4 or
 8. 2. The isolated nucleic acid molecule according to claim 1, comprising the sequence of nucleotides in Sequence I.D. No. 3, from nucleotide number 216 to nucleotide number
 1449. 3. The isolated nucleic acid molecule according to claim 1 wherein said molecule encodes a protein having the amino acid sequence of Sequence I.D. No. 4, from amino acid number 1 to amino acid number
 411. 4. The isolated nucleic acid molecule according to claim 1 wherein said molecule encodes a rat CRF₂ receptor.
 5. The isolated nucleic acid molecule according to claim 1 wherein said molecule encodes a human CRF₂ receptor.
 6. An isolated nucleic acid molecule encoding a mammalian CRF₂ receptor N-terminal extracellular domain, wherein said CRF₂ receptor N-terminal extracellular domain comprises:(a) a nucleic acid molecule which encodes the N-terminal extracellular domain of any one of Sequence I.D. Nos. 2, 4, and 8; or (b) a nucleic acid molecule which hybridizes under conditions of moderate stringency to the complement of a nucleic acid molecule which encodes the N-terminal extracellular domain of any one of Sequence I.D. Nos. 2, 4 or
 8. 7. The isolated nucleic acid molecule according to claim 6 comprising the sequence of nucleotides in Sequence I.D. No. 3, from nucleotide number 216 to nucleotide number
 570. 8. The isolated nucleic acid molecule according to claim 6 wherein said molecule encodes a protein having the amino acid sequence of Sequence I.D. No. 4, from amino acid number 1 to amino acid number
 118. 9. A recombinant expression vector, comprising a promoter operably linked to a nucleic acid molecule according to any one of claims 1-8.
 10. A host cell containing a recombinant expression vector according to claim
 9. 11. A recombinant viral vector capable of directing the expression of a nucleic acid molecule according to any one of claims 1-8 wherein said viral vector is selected from the group consisting of retroviral vectors, adenoviral vectors and herpes simplex virus vectors.
 12. A host cell containing a recombinant expression vector according to claim
 11. 13. An isolated nucleic acid molecule having greater than 85% sequence identity to a nucleic acid molecule encoding an amino acid sequence selected from the group consisting of Sequence ID Nos. 2, 4, or
 8. 